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nasdaq:alny
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Alnylam Pharmaceuticals
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Apr 26th, 2022 12:00AM
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Jul 26th, 2019 12:00AM
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https://www.uspto.gov?id=US11312957-20220426
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Modified iRNA agents
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The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a lipophilic moiety. e.g., cholesterol, is is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.
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11312957
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1. An RNA agent comprising a sense strand and an antisense strand, wherein one or more ribose replacement modification subunit (RRMS) comprising a ligand is incorporated into at least one of said strands, and wherein the RRMS is a cyclic carrier selected from the group consisting of hydroxyproline, piperidine, morpholine, piperazine, and decalin, and wherein the ligand is connected to the RRMS via a tethering moiety containing a C1-C20 alkyl group and having at least one nitrogen atom.
2. The RNA agent of claim 1, wherein the tethering moiety is a C1-C20 alkyl group substituted with a —NHC(O)— group.
3. The RNA agent of claim 2, wherein the tethering moiety is a C1-C10 alkyl group substituted with a —NHC(O)— group.
4. The RNA agent of claim 1, wherein the ligand comprises one or more carbohydrate moieties.
5. The RNA agent of claim 4, wherein the carbohydrate moieties are monosaccharides, disaccharides, trisaccharides, tetrasaccharides, polysaccharides, or combinations thereof.
6. The RNA agent of claim 1, wherein the ligand is selected from the group consisting of a mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, multivalent N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, lactose, galactose, mannose, and combinations thereof.
7. The RNA agent of claim 1, wherein the RRMS subunit is placed within 1, 2, or 3 positions of the 3′ or 5′ end of at least one of said strands.
8. The RNA agent of claim 1, wherein at least two RRMS subunits are incorporated into at least one of said strands.
9. The RNA agent of claim 8, wherein at least three RRMS subunits are incorporated into at least one of said strands.
10. An RNA agent comprising a sense strand and an antisense strand, wherein at least one subunit having a formula (I) is incorporated into at least one of said strands:
wherein:
X is N(CO)R7 or NR7;
Y is NR8, O, S, CR9R10, or absent;
Z is CR11R12 or absent;
each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, ORb, (CH2)nRa, or (CH2)nORb, provided that at least one of R1, R2, R3, R4, R9, and R10 is ORa or ORb and that at least one of R1, R2, R3, R4, R9, and R10 is (CH2)nORa or (CH2)nORb;
each of R5, R6, R11, and R12 is, independently, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14,
R7 is a ligand tethered through a tethering moiety containing a C1-C20 alkyl group and having at least one nitrogen atom;
R8 is C1-C6 alkyl;
R13 is hydroxy, C1-C4 alkoxy, or halo;
R14 is NRcR7,
Ra is H or
Rb is H or
wherein the Strand in each occurrence is independently the sense strand or antisense strand of the iRNA agent;
each of A and C is, independently, O or S;
B is OH, O−, or
Rc is H or C1-C6 alkyl; and
n is 1-4.
11. The RNA agent of claim 10, wherein Y is CR9R10, and Z is absent.
12. The RNA agent of claim 11, wherein R2, R3, R4, R5, R6, and R10 are H; R1 is CH2ORa; and R9 is ORb.
13. The RNA agent of claim 10, wherein the tethering moiety in R7 is a C1-C20 alkyl group substituted with a —NHC(O)— group.
14. The RNA agent of claim 13, wherein the tethering moiety in R7 is a C1-C10 alkyl group substituted with a —NHC(O)— group.
15. The RNA agent of claim 10, wherein the ligand comprises one or more carbohydrate moieties.
16. The RNA agent of claim 15, wherein the carbohydrate moieties are monosaccharides, disaccharides, trisaccharides, tetrasaccharides, polysaccharides, or combinations thereof.
17. The RNA agent of claim 10, wherein the ligand is selected from the group consisting of a mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, multivalent N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, lactose, galactose, mannose, and combinations thereof.
18. The RNA agent of claim 10, wherein the subunit having formula (I) is placed within 1, 2, or 3 positions of the 3′ or 5′ end of at least one of said strands.
19. The RNA agent of claim 10, wherein at least two subunits having formula (I) are incorporated into at least one of said strands.
20. The RNA agent of claim 19, wherein at least three subunits having formula (I) are incorporated into at least one of said strands.
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 15/906,908, filed Feb. 27, 2018, now U.S. Pat. No. 10,676,740, which is a continuation of U.S. application Ser. No. 15/170,693, filed Jun. 1, 2016, now abandoned, which is a continuation of U.S. application Ser. No. 14/281,661, filed May 19, 2014, now U.S. Pat. No. 9,394,540, which is a continuation of U.S. application Ser. No. 13/849,003, filed Mar. 22, 2013, now U.S. Pat. No. 8,796,436, which is a continuation of U.S. application Ser. No. 12/714,298, filed Feb. 26, 2010, now U.S. Pat. No. 8,507,661, which is a continuation of U.S. application Ser. No. 10/916,185, filed Aug. 10, 2004, now U.S. Pat. No. 7,745,608, which is a continuation-in-part of International Application No. PCT/US2004/011829, filed on Apr. 16, 2004, which claims the benefit of U.S. Provisional Application No. 60/493,986, filed on Aug. 8, 2003; U.S. Provisional Application No. 60/494,597, filed on Aug. 11, 2003; U.S. Provisional Application No. 60/506,341, filed on Sep. 26, 2003; U.S. Provisional Application No. 60/518,453, filed on Nov. 7, 2003; U.S. Provisional Application No. 60/463,772, filed on Apr. 17, 2003; U.S. Provisional Application No. 60/465,802, filed on Apr. 25, 2003; U.S. Provisional Application No. 60/469,612, filed on May 9, 2003; U.S. Provisional Application No. 60/510,246, filed on Oct. 9, 2003; U.S. Provisional Application No. 60/510,318, filed on Oct. 10, 2003; U.S. Provisional Application No. 60/503,414, filed on Sep. 15, 2003; U.S. Provisional Application No. 60/465,665, filed on Apr. 25, 2003; International Application No. PCT/US04/07070, filed on Mar. 8, 2004; International Application No. PCT/US2004/10586, filed on Apr. 5, 2004; International Application No. PCT/US2004/11255, filed on Apr. 9, 2004; and International Application No. PCT/US2004/011822, filed on Apr. 16, 2004. The contents of all of these prior applications are hereby incorporated by reference in their entireties.
TECHNICAL FIELD
The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose. The inclusion of such a monomer can allow for modulation of a property of the iRNA agent into which it is incorporated, e.g., by using the non-ribose moiety as a point to which a ligand or other entity, e.g., a lipophilic moiety. e.g., cholesterol, is is directly, or indirectly, tethered. The invention also relates to methods of making and using such modified iRNA agents.
BACKGROUND
RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.
SUMMARY
The inventor has discovered, inter alia, that the ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
The carriers further include (i) at least two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. One of the most preferred moieties is a moiety which promotes entry into a cell, e.g., a lipophilic moiety, e.g., cholesterol. While not wishing to be bound by theory it is believed the attachment of a lipohilic agent increases the lipophilicity of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.
Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).
Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (I) is incorporated into at least one of said strands:
wherein:
X is N(CO)R7, NR7 or CH2;
Y is NR8, O, S, CR9R10, or absent;
Z is CR11R12 or absent;
Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, ORb, (CH2)nORa, or (CH2)nORb, provided that at least one of R1, R2, R3, R4, R9, and R10 is ORa or ORb and that at least one of R1, R2, R3, R4, R9, and R10 is (CH2)nORa, or (CH2)nORb (when the RRMS is terminal, one of R1, R2, R3, R4, R9, and R10 will include Ra and one will include Rb; when the RRMSS is internal, two of R1, R2, R3, R4, R9, and R10 will each include an Rb); further provided that preferably ORa may only be present with (CH2)nORb and (CH2)nORa may only be present with ORb;
Each of R5, R6, R11, and R12 is, independently, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14;
R7 can be a ligand, e.g., R7 can be Rd, or R7 can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd;
R8 is C1-C6 alkyl;
R13 is hydroxy, C1-C4 alkoxy, or halo;
R14 is NRcR7;
Ra is H or:
Rb is H or:
Each of A and C is, independently, O or S;
B is OH, O−, or
Rc is H or C1-C6 alkyl;
Rd is H or a ligand, e.g., a lipophilic ligand, e.g., cholesterol; and
n is 1-4.
Embodiments can include one or more of the following features.
The iRNA agent can be 21 nucleotides in length and there can be a duplex region of about 19 pairs.
The iRNA agent can include a duplex region between 17 and 23 pairs in length.
R1 can be CH2ORa and R3 can be ORb; or R1 can be CH2ORa and R9 can be ORb; or R1 can be CH2ORa and R2 can be ORb.
R1 can be CH2ORb and R3 can be ORb; or R1 can be CH2ORb and R9 can be ORb; or R1 can be CH2ORb and R2 can be ORb; or R1 can be CH2ORb and R3 can be ORa; or R1 can be CH2ORb and R9 can be ORa; or R1 can be CH2ORb and R2 can be ORa.
R1 can be ORa and R3 can be CH2ORb; or R1 can be ORa and R9 can be CH2ORb; or R1 can be ORa and R2 can be CH2ORb.
R1 can be ORb and R3 can be CH2ORb; or R1 can be ORb and R9 can be CH2ORb; or R1 can be ORb and R2 can be CH2ORb; or R1 can be ORb and R3 can be CH2ORa; or R1 can be ORb and R9 can be CH2ORa; or R1 can be ORb and R2 can be CH2ORa.
R3 can be CH2ORa and R9 can be ORb; or R3 can be CH2ORa and R4 can be ORb.
R3 can be CH2ORb and R9 can be ORb; or R3 can be CH2ORb and R4 can be ORb; or R3 can be CH2ORb and R9 can be ORa; or R3 can be CH2ORb and R4 can be ORa.
R3 can be ORb and R9 can be CH2ORa; or R3 can be ORb and R4 can be CH2ORa; or R3 can be ORb and R9 can be CH2ORb; or R3 can be ORb and R4 can be CH2ORb.
R3 can be ORa and R9 can be CH2ORb; or R3 can be ORa and R4 can be CH2ORb.
R9 can be CH2ORa and R10 can be ORb.
R9 can be CH2ORb and R10 can be ORb; or R9 can be CH2ORb and R10 can be ORa.
In a preferred embodiment the ribose is replaced with a pyrroline scaffold or with a 4-hydroxyproline-derived scaffold, and X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent.
R1 and R3 can be cis or R1 and R3 can be trans.
n can be 1.
A can be O or S.
R1 can be (CH2)nORb and R3 can be ORb; or R1 can be (CH2)nORa and R3 can be ORb.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
R1 can be ORb and R3 can be (CH2)nORb; or R1 can be ORb and R3 can be (CH2)nORa; or
R1 can be ORa and R3 can be (CH2)nORb; or R1 can be (CH2)nORb and R9 can be ORa.
R1 and R9 can be cis or R1 and R9 can be trans.
R1 can be ORa and R9 can be (CH2)nORb; or R1 can be (CH2)nORb and R9 can be ORb; or
R1 can be (CH2)nORa and R9 can be ORb; or R1 can be ORb and R9 can be (CH2)nORb; or R1 can be ORb and R9 can be (CH2)nORa.
R3 can be (CH2)nORb and R9 can be ORa; or R3 can be (CH2)nORb and R9 can be ORb; or R3 can be (CH2)nORa and R9 can be ORb; or R3 can be ORa and R9 can be (CH2)nORb; R3 can be ORb and R9 can be (CH2)nORb; or R3 can be ORb and R9 can be (CH2)nORa.
R3 and R9 can be cis or R3 and R9 can be trans.
In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12.
R9 can be (CH2)nORb and R10 can be ORa.
n can be 1 or 2.
R9 can be (CH2)nORb and R10 can be ORb; or R9 can be (CH2)nORa and R10 can be ORb.
A can be O or S.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be selected from a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
R3 can be (CH2)nORb and R4 can be ORa; or R3 can be (CH2)nORb and R4 can be ORb; or
R3 can be (CH2)nORa and R4 can be ORb.
R1 can be (CH2)nORb and R2 can be ORa; or R1 can be (CH2)nORb and R2 can be ORb; or
R1 can be (CH2)nORa and R2 can be ORb.
R3 can be (CH2)nORb and R9 can be ORa.
R3 and R9 can be cis, or R3 and R9 can be trans.
R3 can be (CH2)nORb and R9 can be ORb; or R3 can be (CH2)nORb and R9 can be ORa; or
R3 can be (CH2)nORa and R9 can be ORb.
R1 can be (CH2)nORb and R3 can be ORa.
R1 and R3 can be cis, or R1 and R3 can be trans.
R3 can be ORa and R9 can be (CH2)nORb.
R1 can be ORa and R3 can be (CH2)nORb.
In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R7 or NR7, Y is NR8, and Z is CR11R12.
R1 can be (CH2)nORb and R3 can be ORa.
R1 and R3 can be cis or R1 and R3 can be trans.
n can be 1.
R1 can be (CH2)nORb and R3 can be ORb; or R1 can be (CH2)nORa and R3 can be ORb.
A can be O or S, preferably S.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from the group of a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
R8 can be CH3.
R1 can be ORa and R3 can be (CH2)nORb.
In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R7 or NR7, Y is O, and Z is CR11R12.
R1 can be (CH2)nORb and R3 can be ORa.
R1 and R3 can be cis, or R1 and R3 can be trans.
n can be 1.
R1 can be (CH2)nORb and R3 can be ORb; of R1 can be (CH2)nORa and R3 can be ORb.
A can be O or S.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from the group of a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
R8 can be CH3.
R1 can be ORa and R3 can be (CH2)nORb.
In other preferred embodiments the ribose is replaced with a decalin scaffold, and X is CH2; Y is CR9R10; and Z is CR11R12; and R5 and R11 together are C6 cycloalkyl.
R6 can be C(O)NHR7.
R12 can be hydrogen.
R6 and R12 can be trans.
R3 can be ORa and R9 can be (CH2)nORb.
R3 and R9 can be cis, or R3 and R9 can be trans.
n can be 1 or 2.
R3 can be ORb and R9 can be (CH2)nORb; or R3 can be ORb and R9 can be (CH2)nORa.
A can be O or S.
R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from the group of a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
In other preferred embodiments the ribose is replaced with a decalin/indane scaffold, e.g., X is CH2; Y is CR9R10; and Z is CR11R12; and R5 and R11 together are C5 cycloalkyl.
R6 can be CH3.
R12 can be hydrogen.
R6 and R12 can be trans.
R3 can be ORa and R9 can be (CH2)nORb.
R3 and R9 can be cis, or R3 and R9 can be trans.
n can be 1 or 2.
R3 can be ORb and R9 can be (CH2)nORa; or R3 can be ORb and R9 can be (CH2)nORa.
A can be O or S.
R14 can be N(CH3)R7. R7 can be (CH2)5NHRd or (CH2)5NHRd. Rd can be chosen from the group of a folic acid radical; a cholesterol radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical. Preferably, Rd is a cholesterol radical.
In another aspect, this invention features an iRNA agent comprising a first strand and a second strand, wherein at least one one subunit having a formula (II) is incorporated into at least one of said strands:
X is N(CO)R7 or NR7;
Each of R1 and R2 is, independently, ORa, ORb, (CH2)nORa, or (CH2)nORb, provided that one of R1 and R2 is ORa or ORb and the other is (CH2)nORa or (CH2)nORb (when the RRMS is terminal, one of R1 or R2 will include Ra and one will include Rb; when the RRMSS is internal, both R1 and R2 will each include an Rb); further provided that preferably ORa may only be present with (CH2)nORb and (CH2)nORa may only be present with ORb;
R7 is C1-C20 alkyl substituted with NRcRd;
R8 is C1-C6 alkyl;
R13 is hydroxy, C1-C4 alkoxy, or halo;
R14 is NRcR7;
Ra is:
Rb is
Each of A and C is, independently, O or S;
B is OH, O−, or
Rc is H or C1-C6 alkyl;
Rd is H or a ligand; and
n is 1-4.
Embodiments can include one or more of the features described above.
In a further aspect, this invention features an iRNA agent having a first strand and a second strand, wherein at least one subunit having a formula (I) or formula (II) is incorporated into at least one of said strands.
In one aspect, this invention features an iRNA agent having a first strand and a second strand, wherein at least two subunits having a formula (I) and/or formula (II) are incorporated into at least one of said strands.
In another aspect, this invention provides a method of making an iRNA agent described herein having a first strand and a second strand in which at least one subunit of formula (I) and/or (II) is incorporated in the strands. The method includes contacting the first strand with the second strand.
In a further aspect, this invention provides a method of modulating expression of a target gene, the method includes administering an iRNA agent described herein having a first strand and a second strand in which at least one subunit of formula (I) and/or (II) is incorporated in the strands. to a subject.
In one aspect, this invention features a pharmaceutical composition having an iRNA agent described herein having a first strand and a second strand in which at least one subunit of formula (I) and/or (II) is incorporated in the strands and a pharmaceutically acceptable carrier.
RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.
In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.
In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.
In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”
In other embodiments, an iRNA agent may have an RRMS at the 3′ end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.
In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.
It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.
Other modifications to sugars, bases, or backbones described herein can be incorporated into the iRNA agents.
The iRNA agents can take an architecture or structure described herein. The iRNA agents can be palindromic, or double targeting, as described herein.
The iRNA agents can have a sequence such that a non-cannonical or other than cannonical Watson-Crick structure is formed between two monomers of the iRNA agent or between a strand of the iRNA agent and another sequence, e.g., a target or off-target sequence, as is described herein.
The iRNA agent can be selected to target any of a broad spectrum of genes, including any of the genes described herein.
In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, single strand versus double strand form) described herein. E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In some embodiment the duplex region of the iRNA agent will have, mismatches. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
In addition to the RRMS-containing bases the iRNA agents described herein can include nuclease resistant monomers (NRMs).
In another aspect, the invention features an iRNA agent to which is conjugated a lipophilic moiety, e.g., cholesterol, e.g., by conjugation to an RRMS of an iRNA agent. In a preferred embodiment, the lipophilic moiety enhances entry of the iRNA agent into a cell. In a preferred embodiment, the cell is part of an organism, tissue, or cell line, e.g., a primary cell line, immortalized cell line, or any type of cell line disclosed herein. Thus, the conjugated iRNA agent an be used to silence a target gene in an organism, e.g., a mammal, e.g., a human, or to silence a target gene in a cell line or in cells which are outside an organism.
The lipophilic moiety can be chosen, for example, from the group consisting of a lipid, cholesterol, oleyl, retinyl, cholesteryl residues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. A preferred lipophilic moiety is cholesterol.
The iRNA agent can have a first strand and a second strand, wherein at least one subunit having formula (I) or formula (II) is incorporated into at least one of the strands. The iRNA agent can have one or more of any of the features described herein. For example, when the subunit is of formula (I), Rd can be cholesterol; X can be N(CO)R7 or NR7, Y can be CR9R10, and Z can be absent, and R1 can be (CH2)nORb and R3 can be ORa; X can be N(CO)R7 or NR7, Y can be CR9R10, and Z can be CR11R12, and R9 can be (CH2)nORb and R10 can be ORa; X can be N(CO)R7 or NR7, Y can be NR8, and Z can be CR11R12, and R1 can be (CH2)nORb and R3 can be ORa; X can be CH2; Y can be CR9R10; and Z can be CR11R12, in which R6 can be C(O)NHR7; or X can be CH2; Y can be CR9R10; and Z can be CR11R12, in which R11 or R12 can be C(O) NHR7 or R5 and R11 together can be C5 or C6 cycloalkyl substituted with N(CH3)R7.
In a preferred embodiment, the lipophilic moiety, e.g., a cholesterol, enhances entry of the iRNA agent into a synoviocyte, myocyte, keratinocyte, hepatocyte, leukocyte, endothelial cell (e.g., a kidney cell), B-cell, T-cell, epithelial cell, mesodermal cell, myeloid cell, neural cell, neoplastic cell, mast cell, or fibroblast cell. In certain aspects, a myocyte can be a smooth muscle cell or a cardiac myocyte, a fibroblast cell can be a dermal fibroblast, and a leukocyte can be a monocyte. In another preferred embodiment, the cell can be from an adherent tumor cell line derived from a tissue, such as bladder, lung, breast, cervix, colon, pancreas, prostate, kidney, liver, skin, or nervous system (e.g., central nervous system).
In a preferred embodiment, the iRNA agent targets a protein tyrosine phosphatase (PTP-1B) gene or a MAP kinase gene, such as ERK1, ERK2, JNK1, JNK2, or p38. In a preferred embodiment, these iRNA agents are used to silence genes in a fibroblast cell.
In a preferred embodiment, the iRNA agent targets an MDR, Myc, Myb, c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, v-Myb, cyclin D1, Cyclin D2, cyclin E, CDK4, cdc25A, CDK2, or CDK4 gene. In a preferred embodiment, these iRNA agents are used to silence genes in an epithelial cell or mesodermal cell.
In a preferred embodiment, the iRNA agent targets a G72 or DAAO gene. In a preferred embodiment, these iRNA agents are used to silence genes in a neural cell.
In a preferred embodiment, the iRNA agent targets a gene of the telomerase pathway, such as a TERT or TR/TERC. In a preferred embodiment, these iRNA agents are used to silence genes in a keratinocyte.
In a preferred embodiment, the iRNA agent targets an interleukin gene, such as IL-1, IL-2, IL-5, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. In another preferred embodiment, the iRNA agent targets an interleukin receptor gene, or a chromosomal translocation, such as BCR-ABL, TEL-AML-1, EWS-FLI1, EWS-ERG, TLS-FUS, PAX3-FKHR, or AML-ETO. In a preferred embodiment, these iRNA agents are used to silence genes in a lymphoma or a leukemia cell.
In a preferred embodiment, the iRNA agent targets a GRB2 associated binding protein. In a preferred embodiment, these iRNA agents are used to silence genes in a mast cell.
In a preferred embodiment, the iRNA agent targets a growth factor or growth factor receptor, such as a TGFbeta or TGFbeta Receptor; PDGF or PDGFR; VEGF or VEGFr1, VEGFr2, or VEGFr3; or IGF-1R, DAF-2, or InR. In another preferred embodiment, the iRNA agent targets PRL1, PRL2, PRL3, protein kinase C (PKC), PKC receptor, MDR1, TERT, TR/TERC, cyclin D1, NF-KappaB, REL-A, REL-B, PCNA, CHK-1, c-fos, jun, or BCL-2. In a preferred embodiment, these iRNA agents are used to silence genes in an adherent tumor cell line.
In a preferred embodiment, the iRNA agent targets an exogenous gene of a genetically modified cell. An exogenous gene can be, for example, a viral or bacterial gene that derives from an organism that has invaded or infected the cell, or the exogenous gene can be any gene introduced into the cell by natural or artificial means, such as by a genetic recombination event. An iRNA agent can target a viral gene, for example, such as a hepatitis viral gene (e.g., a gene of an HAV, HBV, or HCV). Alternatively, or in addition, the iRNA agent can silence a reporter gene, such as GFP or beta galatosidase and the like. These iRNA agents can be used to silence exogenous genes in an adherent tumor cell line.
In a preferred embodiment, the iRNA agent to which the lipophilic moiety is conjugated silences at least one gene, e.g., any gene described herein, in any one of a number of cell lines including, but not limited to, a 3T3, DLD2, THP1, Raw264.7, IC21, P388D1, U937, HL60, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, K562, EL4, LRMB, Bcl-1, BC-3, TF1, CTLL-2, C1R, Rat6, VERO, MRC5, CV1, Cos7, RPTE, A10, T24, J82, A549, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, A375, C8161, CCRF-CEM, MCF-7, MDA-MB-231, MOLT, mIMCD-3, NHDF, HeLa, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, LNCaP, HepG2, or U87 cell line. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).
In another aspect, the invention provides, methods of silencing a target gene by providing an iRNA agent to which a lipophilic moiety is conjugated, e.g., a lipophilic conjugated iRNA agent described herein, to a cell. In a preferred embodiment the conjugated iRNA agent an be used to silence a target gene in an organism, e.g., a mammal, e.g., a human, or to silence a target gene in a cell line or in cells which are outside an organism. In the case of a whole organism, the method can be used to silence a gene, e.g., a gene described herein, and treat a condition mediated by the gene. In the case of use on a cell which is not part of an organism, e.g., a primary cell line, secondary cell line, tumor cell line, or transformed or immortalized cell line, the iRNA agent to which a lipophilic moiety is conjugated can be used to silence a gene, e.g., one described herein. Cells which are not part of a whole organism can be used in an initial screen to determine if an iRNA agent is effective in silencing a gene. A test in cells which are not part of a whole organism can be followed by testing the iRNA agent in a whole animal. In preferred embodiments, the iRNA agent which is conjugated to a lipophilic moiety is administered to an organism, or contacted with a cell which is not part of an organism, in the absence of (or in a reduced amount of) other reagents that facilitate or enhance delivery, e.g., a compound which enhances transit through the cell membrane. (A reduced amount can be an amount of such reagent which is reduced in comparison to what would be needed to get an equal amount of nonconjugated iRNA agent into the target cell). E.g., the iRNA agent which is conjugated to a lipophilic moiety is administered to an organism, or contacted with a cell which is not part of an organism, in the absence (or reduced amount) of: an additional lipophilic moiety; a transfection agent, e.g., concentrations of an ion or other substance which substantially alters cell permeability to an iRNA agent; a transfecting agent such as Lipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine2000™, TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.), DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), and the like.
In a preferred embodiment the iRNA agent is suitable for delivery to a cell in vivo, e.g., to a cell in an organism. In another aspect, the iRNA agent is suitable for delivery to a cell in vitro, e.g., to a cell in a cell line.
An iRNA agent to which a lipophilic moiety is attached can target any gene described herein and can be delivered to any cell type described herein, e.g., a cell type in an organism, tissue, or cell line. Delivery of the iRNA agent can be in vivo, e.g., to a cell in an organism, or in vitro, e.g., to a cell in a cell line.
In another aspect, the invention provides compositions of iRNA agents described herein, and in particular compositions of an iRNA agent to which a lipophilic moiety is conjugated, e.g., a lipophilic conjugated iRNA agent described herein. In a preferred embodiment the composition is a pharmaceutically acceptable composition.
In preferred embodiments, the composition, e.g., pharmaceutically acceptable composition, is free of, has a reduced amount of, or is essentially free of other reagents that facilitate or enhance delivery, e.g., compounds which enhance transit through the cell membrane. (A reduced amount can be an amount of such reagent which is reduced in comparison to what would be needed to get an equal amount of nonconjugated iRNA agent into the target cell). E.g., the composition is free of, has a reduced amount of, or is essentially free of: an additional lipophilic moiety; a transfection agent, e.g., concentrations of an ion or other substance which substantially alters cell permeability to an iRNA agent; a transfecting agent such as Lipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™, TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.), DOTAP, DOSPER, Metafectene™ (Biontex, Munich, Germany), and the like.
In a preferred embodiment the composition is suitable for delivery to a cell in vivo, e.g., to a cell in an organism. In another aspect, the iRNA agent is suitable for delivery to a cell in vitro, e.g., to a cell in a cell line.
The RRMS-containing iRNA agents can be used in any of the methods described herein, e.g., to target any of the genes described herein or to treat any of the disorders described herein. They can be incorporated into any of the formulations, modes of delivery, delivery modalities, kits or preparations, e.g., pharmaceutical preparations, described herein. E.g, a kit which includes one or more of the iRNA agents described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.
The methods and compositions of the invention, e.g., the RRSM-containing iRNA agents described herein, can be used with any of the iRNA agents described herein. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.
The methods and compositions of the invention, e.g., the the RRMS-containing iRNA agents described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.
The non-ribose scaffolds, as well as monomers and dimers of the RRMSs described herein are within the invention
An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein, see the section herein entitled RNA Agents. While numerous modified RNAs and nucleoside surrogates are described herein, preferred examples include those which include one or more RRMS. Preferred examples are those which also a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features and advantages of the invention will be apparent from the description and drawings, and from the claims. This application incorporates all cited references, patents, and patent applications by references in their entirety for all purposes.
DESCRIPTION OF DRAWINGS
FIG. 1 a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.
FIG. 2A is a list of substituents that may be present on silicon in OFG1.
FIG. 2B is a list of substituents that may be present on the C2′-orthoester group.
FIG. 3 is list of representative RRMS cyclic carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based RRMSs; panel 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R1 is succinate or phosphoramidate and R2 is H or a conjugate ligand.
FIG. 4 is a general reaction scheme for 3′ conjugation of peptide into iRNA.
FIG. 5 is a general reaction scheme for 5′ conjugation of peptide into iRNA.
FIG. 6 is a general reaction scheme for the synthesis of aza-peptides.
FIG. 7 is a general reaction scheme for the synthesis of N-methyl amino acids and peptides.
FIG. 8 is a general reaction scheme for the synthesis of β-methyl amino acids and Ant and Tat peptides.
FIG. 9 is a general reaction scheme for the synthesis of Ant and Tat oligocarbamates.
FIG. 10 is a a general reaction scheme for the synthesis of Ant and Tat oligoureas.
FIG. 11 is a schematic representation of peptide carriers.
FIG. 12 is a structural representation of base pairing in pseudocomplementary siRNA2.
FIG. 13 is a schematic representation of dual targeting siRNAs designed to target the HCV genome.
FIG. 14 is a schematic representation of pseudocomplementary, bifunctional siRNAs designed to target the HCV genome.
FIG. 15 is a list of control and candidate iRNA agents. SEQ ID NOs for the sense and antisense strands of the duplexes are as follows (sense strand/antisense strand): Duplex #1 (SEQ ID NO. 29/SEQ ID NO. 30), Duplex #2 (SEQ ID NO. 31/SEQ ID NO. 32), Duplex #3 (SEQ ID NO. 33/SEQ ID NO. 34), Duplex #4 (SEQ ID NO. 35/SEQ ID NO. 36), Duplex #5 (SEQ ID NO. 37/SEQ ID NO. 38), Duplex #6 (SEQ ID NO. 55/SEQ ID NO. 56), Duplex #7 (SEQ ID NO. 57/SEQ ID NO. 58), Duplex #8 (SEQ ID NO. 59/SEQ ID NO. 60), Duplex #9 (SEQ ID NO. 45/SEQ ID NO. 46), Duplex #10 (SEQ ID NO. 47/SEQ ID NO. 48), Duplex #11 (SEQ ID NO. 49/SEQ ID NO. 50), and Duplex #12 (SEQ ID NO. 51/SEQ ID NO. 52).
FIG. 16 is a graphical representation of relative cell viability results.
FIG. 17 is a graphical representation of gene silencing activity results.
FIG. 18. is a list of representative cholesterol-tethered RRMS monomers.
FIG. 19 shows LCMS data for a 3′ cholesterol conjugate after PAGE purification.
FIG. 20 is a graphical representation of Luc silencing with no transfection reagent.
FIG. 21 is a denaturing gel analysis of the human serum stability assay for AL-DUP-1000. C is the 4 hour time point for siRNA duplex incubated in PBS buffer alone, OH— is the partial alkaline hydrolysis marker, *s/as represents siRNA duplex containing 5′ end-labeled sense RNA and s/*as represents duplex containing 5′ end-labeled antisense RNA. Samples were incubated in 90% human serum and time points were assayed at 10 seconds, 5 min, 15 min, 30 min, 1 hour, 2 hours and 4 hours. Black lines to the right of bands indicate exonucleolytic degradation fragments and the red lines highlight a few of the endonucleolytic degradation fragment.
FIG. 22A is a denaturing gel analysis of the human serum stability assay for AL-DUP-1393. C is the 4 hour time point for each siRNA duplex incubated in PBS buffer alone, *s/as represents siRNA duplex containing 5′ end-labeled sense RNA and s/*as represents duplex containing 5′ end-labeled antisense RNA. Samples were assayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
FIG. 22B is a denaturing gel analysis of the human serum stability assay for AL-DUP-1329. The lanes are labeled and the experiment was performed as described for FIG. 22A.
FIG. 23 is a denaturing gel analysis of AL-DUP-1036, AL-DUP-13ff, and AL-DUP-1363 (see Table 8 for sequences). Black vertical lines highlight regions where exonuclease cleavage is suppressed, stars indicate sites of strong endonucleolytic cleavage in the antisense strand and weaker endonucleolytic cleavage in the sense strand. C is the 4 hour time point for each siRNA duplex incubated in PBS buffer alone, *s/as represents siRNA duplex containing 5′ end-labeled sense RNA and s/*as represents duplex containing 5′ end-labeled antisense RNA. Samples were assayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
FIG. 24. Human serum stability profile of siRNA duplexes containing cationic modifications. Denaturing gel analysis of AL-DUP-10aa (alkylamino-dT), AL-DUP-1ccc (abasic pyrrolidine cationic), and AL-DUP-1403 (see Table 9 for sequences). Black line highlights region where exonuclease cleavage is suppressed and red star indicates site of strong endonucleolytic cleavage in the antisense strand. C is the 4 hour time point for each siRNA duplex incubated in PBS buffer alone, *s/as represents siRNA duplex containing 5′ end-labeled sense RNA and s/*as represents duplex containing 5′ end-labeled antisense RNA. Samples were assayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
FIG. 25 is a denaturing gel analysis of the human serum stability assay for AL-DUP-1069. The black vertical line highlights the region where exonuclease cleavage is suppressed. C is the 4 hour time point for each siRNA duplex incubated in PBS buffer alone, *s/as represents siRNA duplex containing 5′ end-labeled sense RNA and s/*as represents duplex containing 5′ end-labeled antisense RNA. Samples were assayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
DETAILED DESCRIPTION
Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.
Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA agent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.
Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.
In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.
Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., once per week or once per month.
A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.
Ligand-Conjugated Monomer Subunits and Monomers for Oligonucleotide Synthesis
Definitions
The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.
The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C1-C12 alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.
The term “alkenyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp2 and sp3 carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.
The terms “alkylamino” and “dialkylamino” refer to —NH(alkyl) and —N(alkyl)2 radicals respectively. The term “aralkylamino” refers to a —NH(aralkyl) radical. The term “alkoxy” refers to an —O-alkyl radical, and the terms “cycloalkoxy” and “aralkoxy” refer to an —O-cycloalkyl and O-aralkyl radicals respectively. The term “siloxy” refers to a R3SiO— radical. The term “mercapto” refers to an SH radical. The term “thioalkoxy” refers to an —S-alkyl radical.
The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH2—, —CH2CH2—, and —CH2CH2CH2—. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.
The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom can be substituted. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, anthracenyl, and pyrenyl.
The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom can be substituted. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.
The term “heterocyclyl” refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.
The term “cycloalkenyl” as employed herein includes partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5 to 8 carbons, wherein any ring atom can be substituted. The cycloalkenyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of cycloalkenyl moieties include, but are not limited to cyclohexenyl, cyclohexadienyl, or norbornenyl.
The term “heterocycloalkenyl” refers to a partially saturated, nonaromatic 5-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heterocycloalkenyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond or a common carbon atom (e.g., spiro-fused rings). Examples of heterocycloalkenyl include but are not limited to tetrahydropyridyl and dihydropyran.
The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom can be substituted. The heteroaryl groups herein described may also contain fused rings that share a common carbon-carbon bond.
The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by substituents.
The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO3H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)nalkyl (where n is 0-2), S(O)n aryl (where n is 0-2), S(O)n heteroaryl (where n is 0-2), S(O)n heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned substituents.
The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.
A “protected” moiety refers to a reactive functional group, e.g., a hydroxyl group or an amino group, or a class of molecules, e.g., sugars, having one or more functional groups, in which the reactivity of the functional group is temporarily blocked by the presence of an attached protecting group. Protecting groups useful for the monomers and methods described herein can be found, e.g., in Greene, T. W., Protective Groups in Organic Synthesis (John Wiley and Sons: New York), 1981, which is hereby incorporated by reference.
General
An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting ligand-conjugated monomer subunit is presented as formula (II) below and in the scheme in FIG. 1. The carrier (also referred to in some embodiments as a “linker”) can be a cyclic or acyclic moiety and includes two “backbone attachment points” (e.g., hydroxyl groups) and a ligand. The ligand can be directly attached (e.g., conjugated) to the carrier or indirectly attached (e.g., conjugated) to the carrier by an intervening tether (e.g., an acyclic chain of one or more atoms; or a nucleobase, e.g., a naturally occurring nucleobase optionally having one or more chemical modifications, e.g., an unusual base; or a universal base). The carrier therefore also includes a “ligand or tethering attachment point” for the ligand and tether/tethered ligand, respectively.
The ligand-conjugated monomer subunit may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the ligand-conjugated monomer subunit may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one ligand-conjugated monomer subunit may be present in a RNA molecule, e.g., an iRNA agent. Preferred positions for inclusion of a tethered ligand-conjugated monomer subunits, e.g., one in which a lipophilic moiety, e.g., cholesterol, is tethered to the carrier are at the 3′ terminus, the 5′ terminus, or an internal position of the sense strand.
The modified RNA molecule of formula (II) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding monomer compounds (see, e.g., A, B, and C below and in the scheme in FIG. 1) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.
The monomers, e.g., a ligand-conjugated monomer, generally include two differently functionalized hydroxyl groups (OFG1 and OFG2), which are linked to the carrier molecule (see A below and in FIG. 1), and a ligand/tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C below and in FIG. 1, one hydroxyl group (OFG1) on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG2) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a suitable protecting group, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see variable “R” in A below). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the strand. The wavy line used below (and elsewhere herein) refers to a connection, and can represent a direct bond between the moiety and the attachment point or a tethering molecule which is interposed between the moiety and the attachment point. Directly tethered means the moiety is bound directly to the attachment point. Indirectly tethered means that there is a tether molecule interposed between the attachment point and the moiety.
The (OFG1) protecting group may be selected as desired, e.g., from T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, —OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. In some embodiments, a preferred protecting group is the dimethoxytrityl group. In other embodiments, a preferred protecting group is a silicon-based protecting group having the formula below:
X5′, X5″, and X5″′ can be selected from substituted or unsubstituted alkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy, cycloalkoxy, aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e., R3SiO—, the three “R” groups can be any combination of the above listed groups). X5′, X5″, and X5″′ may all be the same or different; also contemplated is a combination in which two of X5′, X5″, and X5″′ are identical and the third is different. In certain embodiments X5′, X5″, and X5″′ include at least one alkoxy or siloxy groups and may be any one of the groups listed in FIG. 2A, a preferred combination includes X5′, X5″=trimethylsiloxy and X5′=1, 3-(triphenylmethoxy)-2-propoxy or cyclododecyloxy.
Other preferred combinations of X5′, X5″, and X5′ include those that result in OFG1 groups that meet the deprotection and stability criteria delineated below. The group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Rapid removal, i.e., less than one minute, of the silyl group from e.g., a support-bound oligonucleotide is desirable because it can reduce synthesis times and thereby reduce exposure time of the growing oligonucleotide chain to the reagents. Oligonucleotide synthesis can be improved if the silyl protecting group is visible during deprotection, e.g., from the addition of a chromophore silyl substituent.
Selection of silyl protecting groups can be complicated by the competing demands of the essential characteristics of stability and facile removal, and the need to balance these competitive goals. Most substituents that increase stability can also increase the reaction time required for removal of the silyl group, potentially increasing the level of difficulty in removal of the group.
The addition of alkoxy and siloxy substituents to OFG1 silicon-containing protecting groups increases the susceptibility of the protecting groups to fluoride cleavage of the silylether bonds. Increasing the steric bulk of the substituents preserves stability while not decreasing fluoride lability to an equal extent. An appropriate balance of substituents on the silyl group makes a silyl ether a viable nucleoside protecting group.
Candidate OFG1 silicon-containing protecting groups may be tested by exposing a tetrahydrofuran solution of a preferred carrier bearing the candidate OFG1 group to five molar equivalents of tetrahydrofuran at room temperature. The reaction time may be determined by monitoring the disappearance of the starting material by thin layer chromatography.
When the OFG2 in B includes a linker, e.g., a relatively long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG1 is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG2. Subsequent to this operation, OFG1 can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group. R′ can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C1-C10 alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.
OFG2 in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be —C(O)(CH2)qC(O)—, or —C(O)(CH2)qS—, in which q can be 0, 1, 2, 3, or 4; preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5′ silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-stable polystyrene based supports or PEG are preferred.
The ligand/tethering attachment point can be any divalent, trivalent, tetravalent, pentavalent or hexavalent atom. In some embodiments, ligand/tethering attachment point can be a carbon, oxygen, nitrogen or sulfur atom. For example, a ligand/tethering attachment point precursor functional group can have a nucleophilic heteroatom, e.g., —SH, —NH2, secondary amino, ONH2, or NH2NH2. As another example, the ligand/tethering attachment point precursor functional group can be an olefin, e.g., —CH═CH2, and the precursor functional group can be attached to a ligand, a tether, or tethered ligand using, e.g., transition metal catalyzed carbon-carbon (for example olefin metathesis) processes or cycloadditions (e.g., Diels-Alder). As a further example, the ligand/tethering attachment point precursor functional group can be an electrophilic moiety, e.g., an aldehyde. When the carrier is a cyclic carrier, the ligand/tethering attachment point can be an endocyclic atom (i.e., a constituent atom in the cyclic moiety, e.g., a nitrogen atom) or an exocyclic atom (i.e., an atom or group of atoms attached to a constituent atom in the cyclic moiety).
The carrier can be any organic molecule containing attachment points for OFG1, OFG2, and the ligand. In certain embodiments, carrier is a cyclic molecule and may contain heteroatoms (e.g., O, N or S). E.g., carrier molecules may include aryl (e.g., benzene, biphenyl, etc.), cycloalkyl (e.g., cyclohexane, cis or trans decalin, etc.), or heterocyclyl (piperazine, pyrrolidine, etc.). In other embodiments, the carrier can be an acyclic moiety, e.g., based on serinol. Any of the above cyclic systems may include substituents in addition to OFG1, OFG2, and the ligand.
Sugar-Based Monomers
In some embodiments, the carrier molecule is an oxygen containing heterocycle. Preferably the carrier is a ribose sugar as shown in structure LCM-I. In this embodiment, the monomer, e.g., a ligand-conjugated monomer is a nucleoside.
“B” represents a nucleobase, e.g., a naturally occurring nucleobase optionally having one or more chemical modifications, e.g., and unusual base; or a universal base.
As used herein, an “unusual” nucleobase can include any one of the following:
2-methyladeninyl,
N6-methyladeninyl,
2-methylthio-N6-methyladeninyl,
N6-isopentenyladeninyl,
2-methylthio-N6-isopentenyladeninyl,
N6-(cis-hydroxyisopentenyl)adeninyl,
2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,
N6-glycinylcarbamoyladeninyl,
N6-threonylcarbamoyladeninyl,
2-methylthio-N6-threonyl carbamoyladeninyl,
N6-methyl-N6-threonylcarbamoyladeninyl,
N6-hydroxynorvalyl carbamoyladeninyl,
2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,
N6,N6-dimethyladeninyl,
3-methylcytosinyl,
5-methylcytosinyl,
2-thiocytosinyl,
5-formylcytosinyl,
N4-methylcytosinyl,
5-hydroxymethylcytosinyl,
1-methylguaninyl,
N2-methylguaninyl,
7-methylguaninyl,
N2,N2-dimethylguaninyl,
N2,7-dimethylguaninyl,
N2,N2,7-trimethylguaninyl,
1-methylguaninyl,
7-cyano-7-deazaguaninyl,
7-aminomethyl-7-deazaguaninyl,
pseudouracilyl,
dihydrouracilyl,
5-methyluracilyl,
1-methylpseudouracilyl,
2-thiouracilyl,
4-thiouracilyl,
2-thiothyminyl
5-methyl-2-thiouracilyl,
3-(3-amino-3-carboxypropyl)uracilyl,
5-hydroxyuracilyl,
5-methoxyuracilyl,
uracilyl 5-oxyacetic acid,
uracilyl 5-oxyacetic acid methyl ester,
5-(carboxyhydroxymethyl)uracilyl,
5-(carboxyhydroxymethyl)uracilyl methyl ester,
5-methoxycarbonylmethyluracilyl,
5-methoxy carbonyl methyl-2-thiouracilyl,
5-aminomethyl-2-thiouracilyl,
5-methylaminomethyluracilyl,
5-methylaminomethyl-2-thiouracilyl,
5-methylaminomethyl-2-selenouracilyl,
5-carbamoylmethyluracilyl,
5-carboxymethylaminomethyluracilyl,
5-carboxymethylaminomethyl-2-thiouracilyl,
3-methyluracilyl,
1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,
5-carboxymethyluracilyl,
5-methyldihydrouracilyl, or
3-methylpseudouracilyl.
A universal base can form base pairs with each of the natural DNA/RNA bases, exhibiting relatively little discrimination between them. In general, the universal bases are non-hydrogen bonding, hydrophobic, aromatic moieties which can stabilize e.g., duplex RNA or RNA-like molecules, via stacking interactions. A universal base can also include hydrogen bonding substituents. As used herein, a “universal base” can include anthracenes, pyrenes or any one of the following:
In some embodiments, B can form part of a tether that connects a ligand to the carrier. For example, the tether can be B—CH═CH—C(O)NH—(CH2)5—NHC(O)-LIGAND. In a preferred embodiment, the double bond is trans, and the ligand is a substituted or unsubstituted cholesterolyl radical (e.g., attached through the D-ring side chain or the C-3 hydroxyl); an aralkyl moiety having at least one sterogenic center and at least one substituent on the aryl portion of the aralkyl group; or a nucleobase. In certain embodiments, B, in the tether described above, is uracilyl or a universal base, e.g., an aryl moiety, e.g., phenyl, optionally having additional substituents, e.g., one or more fluoro groups. B can be substituted at any atom with the remainder of the tether.
X2 can include “oxy” or “deoxy” substituents in place of the 2′-OH; or be a ligand or a tethered ligand.
Examples of “oxy”-substituents include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, sugar, or protecting group); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-PROTECTED AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH2)nPROTECTED AMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino), and orthoester. Amine protecting groups can include formyl, amido, benzyl, allyl, etc.
Preferred orthoesters have the general formula J. The groups R31 and R32 may be the same or different and can be any combination of the groups listed in FIG. 2B. A preferred orthoester is the “ACE” group, shown below as structure K.
“Deoxy” substituents include hydrogen (i.e. deoxyribose sugars); halo (e.g., fluoro); protected amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid in which all amino are protected); fully protected polyamino (e.g., NH(CH2CH2NH)nCH2CH2-AMINE, wherein AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino and all amino groups are protected), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., a protected amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.
X3 is as described for OFG2 above.
PG can be a triarylmethyl group (e.g., a dimethoxytrityl group) or Si(X5′)(X5″)(X5″′) in which (X5′), (X5″), and (X5″′) are as described elsewhere.
Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated Monomers (Cyclic)
Cyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds. Preferred carriers have the general formula (LCM-2) provided below (In that structure preferred backbone attachment points can be chosen from R1 or R2; R3 or R4; or R9 and R10 if Y is CR9R10 (two positions are chosen to give two backbone attachment points, e.g., R1 and R4, or R4 and R9)). Preferred tethering attachment points include R7; R5 or R6 when X is CH2. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., R1 or R2; R3 or R4; or R9 or R10 (when Y is CR9R10), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be —CH2—, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)
in which,
X is N(CO)R7, NR7 or CH2;
Y is NR8, O, S, CR9R10;
Z is CR11R12 or absent;
Each of R1, R2, R3, R4, R9, and R10 is, independently, H, ORa, or (CH2)nORb, provided that at least two of R1, R2, R3, R4, R9, and R10 are ORa and/or (CH2)nORb;
Each of R5, R6, R11, and R12 is, independently, a ligand, H, C1-C6 alkyl optionally substituted with 1-3 R13, or C(O)NHR7; or R5 and R11 together are C3-C8 cycloalkyl optionally substituted with R14;
R7 can be a ligand, e.g., R7 can be Rd, or R7 can be a ligand tethered indirectly to the carrier, e.g., through a tethering moiety, e.g., C1-C20 alkyl substituted with NRcRd; or C1-C20 alkyl substituted with NHC(O)Rd;
R8 is H or C1-C6 alkyl;
R13 is hydroxy, C1-C4 alkoxy, or halo;
R14 is NRcR7;
R15 is C1-C6 alkyl optionally substituted with cyano, or C2-C6 alkenyl;
R16 is C1-C10 alkyl;
R17 is a liquid or solid phase support reagent;
L is —C(O)(CH2)qC(O)—, or —C(O)(CH2)qS—;
Ra is a protecting group, e.g., CAr3; (e.g., a dimethoxytrityl group) or Si(X5′)(X5″)(X5″′) in which (X5′), (X5″), and (X5″′) are as described elsewhere.
Rb is P(O)(O−)H, P(OR15)N(R16)2 or L-R17;
Rc is H or C1-C6 alkyl;
Rd is H or a ligand;
Each Ar is, independently, C6-C10 aryl optionally substituted with C1-C4 alkoxy;
n is 1-4; and q is 0-4.
Exemplary carriers include those in which, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent; or X is N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is NR8, and Z is CR11R12; or X is N(CO)R7 or NR7, Y is O, and Z is CR11R12; or X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C6 cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C5 cycloalkyl (H, z=1).
In certain embodiments, the carrier may be based on the pyrroline ring system or the 4-hydroxyproline ring system, e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is absent (D). OFG1 is preferably attached to a primary carbon, e.g., an exocyclic alkylene
group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (—CH2OFG1 in D). OFG2 is preferably attached directly to one of the carbons in the five-membered ring (—OFG2 in D). For the pyrroline-based carriers, —CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or —CH2OFG1 may be attached to C-3 and OFG2 may be attached to C-4. In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above-referenced carbons. For the 3-hydroxyproline-based carriers, —CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-4. The pyrroline- and 4-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen. Preferred examples of carrier D include the following:
In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R7 or NR7, Y is CR9R10, and Z is CR11R12. OFG1 is preferably
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [—(CH2)nOFG1 in E]. OFG2 is preferably attached directly to one of the carbons in the six-membered ring (—OFG2 in E). —(CH2)nOFG1 and OFG2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g.,
—(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; —(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; —(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or —(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen.
In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R7 or NR7, Y is NR8, and Z is CR11R12, or the morpholine ring system (G), e.g., X is N(CO)R7 or NR7, Y is O, and Z is CR11R12. OFG1 is preferably
attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (—CH2OFG1 in F or G). OFG2 is preferably attached directly to one of the carbons in the six-membered rings (—OFG2 in F or G). For both F and G, —CH2OFG1 may be attached to C-2 and OFG2 may be attached to C-3; or vice versa. In certain embodiments, CH2OFG1 and OFG2 may be geminally substituted to one of the above-referenced carbons. The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH2OFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). R″′ can be, e.g., C1-C6 alkyl, preferably CH3. The tethering attachment point is preferably nitrogen in both F and G.
In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C6 cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH2; Y is CR9R10; Z is CR11R12, and R5 and R11 together form C5 cycloalkyl (H, z=1). OFG1 is preferably attached to a primary carbon,
e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [—(CH2)nOFG1 in H]. OFG2 is preferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG2 in H). —(CH2)nOFG1 and OFG2 may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, —(CH2)nOFG1 and OFG2 may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH2)nOFG1 may be attached to C-2 and OFG2 may be attached to C-3; —(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-2; —(CH2)nOFG1 may be attached to C-3 and OFG2 may be attached to C-4; or —(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-3; —(CH2)nOFG1 may be attached to C-4 and OFG2 may be attached to C-5; or —(CH2)nOFG1 may be attached to C-5 and OFG2 may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.
Other carriers may include those based on 3-hydroxyproline (J). Thus, —(CH2)nOFG1 and OFG2 may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers
and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included (e.g., the centers bearing CH2OFG1 and OFG2 can both have the R configuration; or both have the S configuration; or one center can have the R configuration and the other center can have the S configuration and vice versa). The tethering attachment point is preferably nitrogen.
Representative cyclic, sugar replacement-based carriers are shown in FIG. 3.
Sugar Replacement-Based Monomers (Acyclic)
Acyclic sugar replacement-based monomers, e.g., sugar replacement-based ligand-conjugated monomers, are also referred to herein as ribose replacement monomer subunit (RRMS) monomer compounds. Preferred acyclic carriers can have formula LCM-3 or LCM-4 below.
In some embodiments, each of x, y, and z can be, independently of one another, 0, 1, 2, or 3. In formula LCM-3, when y and z are different, then the tertiary carbon can have either the R or S configuration. In preferred embodiments, x is zero and y and z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z are each 1 in formula LCM-3. Each of formula LCM-3 or LCM-4 below can optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl.
Tethers
In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at a tethering attachment point (TAP) and may include any C1-C10 carbon-containing moiety, (e.g. C1-C75, C1-C50, C1-C20, C1-C10; C1, C2, C3, C4, C5, C6, C7, C8, C9, or C10), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino or amido (NHC(O)—) group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP-(CH2)nNH—; TAP-C(O)(CH2)nNH—; TAP-NR″″(CH2)nNH—, TAP-C(O)—(CH2)n—C(O)—; TAP-C(O)—(CH2)n—C(O)O—; TAP-C(O)—O—; TAP-C(O)—(CH2)n—NH—C(O)—; TAP-C(O)—(CH2)n—; TAP-C(O)—NH—; TAP-C(O)—; TAP-(CH2)n—C(O)—; TAP-(CH2)n—C(O)O—; TAP-(CH2)n—; or TAP-(CH2)n—NH—C(O)—; in which n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and R″″ is C1-C6 alkyl. Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., —ONH2, or hydrazino group, —NHNH2. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH2)nNH(LIGAND); TAP-C(O)(CH2)nNH(LIGAND); TAP-NR″″(CH2)nNH(LIGAND); TAP-(CH2)nONH(LIGAND); TAP-C(O)(CH2)nONH(LIGAND); TAP-NR″″(CH2)nONH(LIGAND); TAP-(CH2)nNHNH2(LIGAND), TAP-C(O)(CH2)nNHNH2(LIGAND); TAP-NR″″(CH2)nNHNH2(LIGAND); TAP-C(O)—(CH2)n—C(O)(LIGAND); TAP-C(O)—(CH2)n—C(O)O(LIGAND); TAP-C(O)—O(LIGAND); TAP-C(O)—(CH2)n—NH—C(O)(LIGAND); TAP-C(O)—(CH2)n(LIGAND); TAP-C(O)—NH(LIGAND); TAP-C(O)(LIGAND); TAP-(CH2)n—C(O) (LIGAND); TAP-(CH2)n—C(O)O(LIGAND); TAP-(CH2)n(LIGAND);_or TAP-(CH2)n—NH—C(O)(LIGAND). In some embodiments, amino terminated tethers (e.g., NH2, ONH2, NH2NH2) can form an imino bond (i.e., C═N) with the ligand. In some embodiments, amino terminated tethers (e.g., NH2, ONH2, NH2NH2) can acylated, e.g., with C(O)CF3.
In some embodiments, the tether can terminate with a mercapto group (i.e., SH) or an olefin (e.g., CH═CH2). For example, the tether can be TAP-(CH2)n—SH, TAP-C(O)(CH2)nSH, TAP-(CH2)n—(CH═CH2), or TAP-C(O)(CH2)n(CH═CH2), in which n can be as described elsewhere. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. The double bond can be cis or trans or E or Z.
In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP-(CH2)nCHO; TAP-C(O)(CH2)nCHO; or TAP-NR″″(CH2)nCHO, in which n is 1-6 and R″″ is C1-C6 alkyl; or TAP-(CH2)nC(O)ONHS; TAP-C(O)(CH2) nC(O)ONHS; or TAP-NR″″(CH2)nC(O)ONHS, in which n is 1-6 and R″″ is C1-C6 alkyl; TAP-(CH2)nC(O)OC6F5; TAP-C(O)(CH2) nC(O) OC6F5; or TAP-NR″″(CH2)nC(O) OC6F5, in which n is 1-11 and R″″ is C1-C6 alkyl; or —(CH2)nCH2LG; TAP-C(O)(CH2)nCH2LG; or TAP-NR″″(CH2)nCH2LG, in which n can be as described elsewhere and R″″ is C1-C6 alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.
In other embodiments, it can be desirable for the monomer to include a phthalimido group (K) at the terminal position of the tether.
In other embodiments, other protected amino groups can be at the terminal position of the tether, e.g., alloc, monomethoxy trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the aryl portion can be ortho-nitrophenyl or ortho, para-dinitrophenyl).
Any of the tethers described herein may further include one or more additional linking groups, e.g., —O—(CH2)n—, —(CH2)n—SS—, —(CH2)n—, or —(CH═CH)—.
Tethered Ligands
A wide variety of entities, e.g., ligands, can be tethered to an iRNA agent, e.g., to the carrier of a ligand-conjugated monomer subunit. Examples are described below in the context of a ligand-conjugated monomer subunit but that is only preferred, entities can be coupled at other points to an iRNA agent.
Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the ligand-conjugated monomer when the ligand-conjugated monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” ligand-conjugated monomer subunit after a “precursor” ligand-conjugated monomer subunit has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether, e.g., TAP-(CH2)nNH2 may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer subunit into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor ligand-conjugated monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor ligand-conjugated monomer subunit tether.
In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.
Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics.
Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic moieties, e.g., cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters and ethers thereof, e.g., C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.
In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and petomimetics can target cancer cells, in particular cells that exhibit an Ivϑ3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the Iv—ϑ3 integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of a monomore derivitzed with a ligand which targets to the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, for example).
TABLE 1
Exemplary Cell Permeation Peptides
Cell Permeation
Peptide
Amino acid Sequence
Reference
Penetratin
RQIKIWFQNRRMKWKK (SEQ ID NO: 1)
Derossi et al., J.
Biol. Chem. 269: 10444,
1994
Tat fragment
GRKKRRQRRRPPQC (SEQ ID NO: 2)
Vives et al., J.
(48-60)
Biol. Chem., 272: 16010,
1997
Signal Sequence-
GALFLGWLGAAGSTMGAWSQPKKKRKV
Chaloin et al.,
based peptide
(SEQ ID NO: 3)
Biochem. Biophys. Res.
Commun., 243: 601,
1998
PVEC
LLIILRRRIRKQAHAHSK (SEQ ID NO: 4)
Elmquist et al.,
Exp. Cell Res., 269: 237,
2001
Transportan
GWTLNSAGYLLKINLKALAALAKKIL
Pooga et al.,
(SEQ ID NO: 5)
FASEB J., 12: 67, 1998
Amphiphilic
KLALKLALKALKAALKLA (SEQ ID NO: 6)
Oehlke et al.,
model peptide
Mol. Ther., 2: 339, 2000
Arg9
RRRRRRRRR (SEQ ID NO: 7)
Mitchell et al., J. Pept.
Res., 56: 318, 2000
Bacterial cell
KFFKFFKFFK (SEQ ID NO: 8)
wall permeating
LL-37
LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN
LVPRTES (SEQ ID NO: 9)
Cecropin P1
SWLSKTAKKLENSAKKRISEGIAIAIQGGP
R (SEQ ID NO: 10)
α-defensin
ACYCRIPACIAGERRYGTCIYQGRLWAFC
C (SEQ ID NO: 11)
b-defensin
DHYNCVSSGGQCLYSACPIFTKIQGTCYR
GKAKCCK (SEQ ID NO: 12)
Bactenecin
RKCRIVVIRVCR (SEQ ID NO: 13)
PR-39
RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP
RFPPRFPGKR-NH2 (SEQ ID NO: 14)
Indolicidin
ILPWKWPWWPWRR-NH2 (SEQ ID NO: 15)
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 16). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 17)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 18)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 19)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing αVß3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an Ivϑ3 integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the Iv—ϑ3 integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, 3-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
In one embodiment, a targeting peptide tethered to an ligand-conjugated monomer can be an amphipathic α-helical peptide. Exemplary amphipathic α-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i+3, or i+4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.
Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
In some embodiments, the ligand can be any of the nucleobases described herein.
In some embodiments, the ligand can be a substituted amine, e.g. dimethylamino. In certain embodiments the substituted amine can be rendered cationic, e.g., by quaternization, e.g., protonation or alkylation. In certain embodiments, the substituted amine can be at the terminal position of a relatively hydrophobic chain, e.g., an alkylene chain.
In some embodiments, the ligand can be one of the following triterpenes:
In some embodiments, the ligand can be substituted or unsubstituted cholesterol, or a stereoisomer thereof or one of the following steroids:
Methods for Making iRNA Agents
A listing of ribonucleosides containing the unusual bases described herein are described in “The RNA Modification Database” maintained by Pamela F. Crain, Jef Rozenski and James A. McCloskey; Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 84112, USA (RNAmods@lib.med.utah.edu)
The 5′ silyl protecting group can be used in conjunction with acid labile orthoesters at the 2′ position of ribonucleosides to synthesize oligonucleotides via phosphoramidite chemistry. Final deprotection conditions are known not to significantly degrade RNA products. Functional groups on the unusual and universal bases are blocked during oligonucleotide synthesis with protecting groups that are compatible with the operations being performed that are described herein. All syntheses can be can be conducted in any automated or manual synthesizer on large, medium, or small scale. The syntheses may also be carried out in multiple well plates or glass slides.
The 5′-O-silyl group can be removed via exposure to fluoride ions, which can include any source of fluoride ion, e.g., those salts containing fluoride ion paired with inorganic counterions e.g., cesium fluoride and potassium fluoride or those salts containing fluoride ion paired with an organic counterion, e.g., a tetraalkylammonium fluoride. A crown ether catalyst can be utilized in combination with the inorganic fluoride in the deprotection reaction. Preferred fluoride ion source are tetrabutylammonium fluoride or aminehydrofluorides (e.g., combining aqueous HF with triethylamine in a dipolar aprotic solvent, e.g., dimethylformamide).
The choice of protecting groups for use on the phosphite triesters and phosphotriesters can alter the stability of the triesters towards fluoride. Methyl protection of the phosphotriester or phosphitetriester can stabilize the linkage against fluoride ions and improve process yields.
Since ribonucleosides have a reactive 2′ hydroxyl substituent, it can be desirable to protect the reactive 2′ position in RNA with a protecting group that is compatible with a 5′-O-silyl protecting group, e.g. one stable to fluoride. Orthoesters meet this criterion and can be readily removed in a final acid deprotection step that can result in minimal RNA degradation.
Tetrazole catalysts can be used in the standard phosphoramidite coupling reaction. Preferred catalysts include e.g. tetrazole, S-ethyl-tetrazole, p-nitrophenyltetrazole.
The general process is as follows. Nucleosides are suitably protected and functionalized for use in solid-phase or solution-phase synthesis of RNA oligonucleotides. The 2′-hydroxyl group in a ribonucleotide can be modified using a tris orthoester reagent. The 2′-hydroxyl can be modified to yield a 2′-O-orthoester nucleoside by reacting the ribonucleoside with the tris orthoester reagent in the presence of an acidic catalyst, e.g., pyridinium p-toluene sulfonate. This reaction is known to those skilled in the art. The product can then be subjected to further protecting group reactions (e.g., 5′-O-silylation) and functionalizations (e.g., 3′-O-phosphitylation) to produce a desired reagent (e.g., nucleoside phosphoramidite) for incorporation within an oligonucleotide or polymer by reactions known to those skilled in the art.
Preferred orthoesters include those comprising ethylene glycol ligands which are protected with acyl or ester protecting groups. Specifically, the preferred acyl group is acetyl. The nucleoside reagents may then be used by those skilled in the art to synthesize RNA oligonucleotides on commercially available synthesizer instruments, e.g. Gene Assembler Plus (Pharmacia), 380B (Applied Biosystems). Following synthesis (either solution-phase or solid-phase) of an oligonucleotide or polymer, the product can be subjected to one or more reactions using non-acidic reagents. One of these reactions may be strong basic conditions, for example, 40% methylamine in water for 10 minutes at 55.degree. C., which will remove the acyl protecting groups from the ethylene glycol ligands but leave the orthoester moiety attached. The resultant orthoester may be left attached when the polymer or oligonucleotide is used in subsequent applications, or it may be removed in a final mildly-acidic reaction, for example, 10 minutes at 55.degree. C. in 50 mM acetic acid, pH 3.0, followed by addition of equal volume of 150 mM TRIS buffer for 10 minutes at 55.degree. C.
Universal bases are described in “Survey and Summary: The Applications of Universal DNA base analogues” Loakes, D., Nucleic Acid Research 2001, 29, 2437, which is incorporated by reference in its entirety. Specific examples are described in the following: Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926; Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632; Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192; Moran, S. Ren, R. X.-F.; Rumney I V, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804; Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440; Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632; McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376; Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13.
As discussed above, the monomers and methods described herein can be used in the preparation of modified RNA molecules, or polymeric molecules comprising any combination of monomer compounds described herein and/or natural or modified ribonucleotides in which one or more subunits contain an unusual or universal base. Modified RNA molecules include e.g. those molecules containing a chemically or stereochemically modified nucleoside (e.g., having one or more backbone modifications, e.g., phosphorothioate or P-alkyl; having one or more sugar modifications, e.g., 2′-OCH3 or 2′-F; and/or having one or more base modifications, e.g., 5-alkylamino or 5-allylamino) or a nucleoside surrogate.
Coupling of 5′-hydroxyl groups with phosphoramidites forms phosphite ester intermediates, which in turn are oxidized e.g., with iodine, to the phosphate diester. Alternatively, the phosphites may be treated with e.g., sulfur, selenium, amino, and boron reagents to form modified phosphate backbones. Linkages between the monomers described herein and a nucleoside or oligonucleotide chain can also be treated with iodine, sulfur, selenium, amino, and boron reagents to form unmodified and modified phosphate backbones respectively. Similarly, the monomers described herein may be coupled with nucleosides or oligonucleotides containing any of the modifications or nucleoside surrogates described herein.
The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001. Exemplary methods are shown in FIGS. 4 and 5.
In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the α-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a “carbonyl donor,” e.g., phenylchloroformate. A general azapeptide synthesis is shown in FIG. 6.
In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an ligand-conjugated monomer) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide. An exemplary synthesis is shown in FIG. 7.
In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to a ligand-conjugated monomer) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. β-peptides are resistant to degradation by proteolytic enzymes. β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide. An exemplary synthesis is shown in FIG. 8.
In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to a ligand-conjugated monomer) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate. An exemplary synthesis is shown in FIG. 9.
In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to a ligand-conjugated monomer) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate). An exemplary synthesis is shown in FIG. 10.
The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, ligand-conjugated monomers occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.
A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.
A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes. A schematic of preferred carriers is shown in FIG. 11.
The protected monomer compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Other synthetic chemistry transformations, protecting groups (e.g., for hydroxyl, amino, etc. present on the bases) and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
The protected monomer compounds of this invention may contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of these compounds are expressly included in the present invention. The compounds described herein can also contain linkages (e.g., carbon-carbon bonds, carbon-nitrogen bonds, e.g., amides) or substituents that can restrict bond rotation, e.g. restriction resulting from the presence of a ring or double bond. Accordingly, all cis/trans, E/Z isomers, and rotational isomers (rotamers) are expressly included herein. The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.
Representative ligand-conjugated monomers and typical syntheses for preparing ligand-conjugated monomers and related compounds described herein are provided below. As discussed elsewhere, protecting groups for ligand-conjugated monomer hydroxyl groups, e.g., OFG1, include but are not limited to the dimethoxytrityl group (DMT). For example, it can be desirable in some embodiments to use silicon-based protecting groups as a protecting group for OFG1. Silicon-based protecting groups can therefore be used in conjunction with or in place of the DMT group as necessary or desired. Thus, the ligand-conjugated monomers and syntheses delineated below, which feature the DMT protecting group as a protecting group for OFG1, is not to be construed as limiting in any way to the invention.
Synthesis of Pyrroline Carrier
Synthesis of 5′-Labelled siRNA
25 & 26 can be used for 3′, 5′-conjugation respectively.
Synthesis of Pthalimido Derivative
30 and 31 can be converted to similar derivatives as shown in schemes 2-4 for 3′ and 5′ conjugation of siRNA
Synthesis of Thalimido Derivative
40 and 41 can be converted to similar derivatives as shown in schemes 2-4 for 3′ and 5′ conjugation of siRNA
Synthesis of N-Alkyl Pyrroline Derivatives
Intermediates 50 and 51 can be converted to analogs which could be conjugated with siRNA using similar reactions
Piperidine Series Ligands:
Similar to pyrroline series piperidine series can be synthesised
Piperidine Series Ligands:
Similar to pyrroline series piperidine series can be synthesised
Hydroxy Proline Series Linkers:
From commercially available cis-3-hydroxy proline and (s)-pyrrolidone carboxylate
Phthalimide Derivative to Stabilise siRNA
4-Hydroxy Proline Derivatives
Phthalimido Derivatives
Synthesis of 6-Membered Linker
Similar reaction can be carried out with 2-piperidone and 3-piperidone
Linkers from 4-Piperidone
Linkers from 3-Piperidone
Linkers from 2-Piperidone
Conjugation Through Decalin System
Conjugates from Decalin System:
Decalin Linker from Wieland-Miescher Ketone
Conjugates from Wieland-Miescher Ketone
Synthesis of Pyrroline Linker
Solid Phase Synthesis and Post-Synthesis Conjugation:
Exemplary Ligand Conjugated Monomers
LCM-E.g.—
Targeting
The iRNA agents of the invention are particularly useful when targeted to the liver. The chemical modifications described herein can be combined with the compounds and methods described in U.S. Provisional Application 60/462,097, filed on Apr. 9, 2003, which is hereby incorporated by reference; and U.S. Provisional Application 60/461,915, filed on Apr. 10, 2003, which is hereby incorporated by reference. For example, an iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver, e.g., a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.
An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.
Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to a non-kidney tissue, such as the liver.
An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the α-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase; glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.
A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, can be useful. These agents target, for example, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.
The iRNA agents of the invention are particularly useful when targeted to the kidney. The chemical modifications described herein can be combined with the compounds and methods described in U.S. Provisional Application 60/460,783, filed on Apr. 3, 2003, which is hereby incorporated by reference; and 60/503,414, filed on Sep. 15, 2003, which is hereby incorporated by reference. An iRNA agent can be targeted to the kidney by incorporation of an RRMS containing a ligand that targets the kidney.
An iRNA agent targeted to the kidney by an RRMS targeting moiety described herein can target a gene expressed in the kidney.
Ligands on RRMSs can include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.
Conjugation with a Lipophilic Moiety which Promotes Entry into Cells
RNAi agents can be modified so as to enhance entry into cells, e.g., by conjugation with a lipophilic moiety. A lipophilic moiety can be attached to an RNAi agent in a number of ways but a preferred mode of attachment is by attachment to an RRMS, e.g., pyrroline-based RRMS. The lipohilic moiety can be attached at the N atom of a pyrroline-based RRMS. Examples of lipophilic moieties include cholesterols, lipid, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Cholesterol is a particularly preferred example.
The lipohilic moiety can be attached at the 3′ terminus, the 5′terminus, or internally, preferably on the sense strand. The lipohilic moiety can be attached to an RRMS, e.g., a pyrroline-based RRMS which is at the 3′ terminus, the 5′terminus, or internal, in the sense strand. The attachment can be direct or through a tethering molecule. Tethers, spacers or linkers discussed herein can be used to attach the moiety to the RRMS.
An iRNA agent to which one or more lipophilic (e.g., cholesterol) molecules is conjugated (referred to herein as an “iRNA-lipophilic conjugate”) can be delivered in vivo, e.g., to a cell, such as a cell of a tissue in a subject, such as a mammalian subject (e.g., a human or mouse). Alternatively, or in addition, the iRNA agent can be delivered in vitro, e.g., to a cell in a cell line. Cell lines can be, for example, from a vertebrate organism, such as a mammal (e.g., a human or a mouse). Delivery of an iRNA-cholesterol conjugate to a cell line can be in the absence of other transfection reagents. For example, delivery of an iRNA-lipophilic conjugate to a cell can be in the absence of, or optionally, in the presence of, Lipofectamine™ (Invitrogen, Carlsbad, Calif.), Lipofectamine 2000™, TransIT-TKO™ (Mirus, Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.), polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.), DOTAP, DOSPER, or Metafectene™ (Biontex, Munich, Germany), or another transfection reagent. Exemplary cell lines can be provided by the American Type Culture Collection (ATCC) (Manassas, Va.). An iRNA-lipophilic conjugate can be delivered to a cell line, such as any cell line described herein, to target a specific gene for downregulation.
In one example, an iRNA-lipophilic conjugate can be delivered to a primary cell line, e.g., a synoviocyte (such as type B), cardiac myocyte, keratinocyte, hepatocyte, smooth muscle cell, endothelial cell, or dermal fibroblast cell line.
In another example, an iRNA-lipophilic conjugate can be delivered to monocyte, or myeloid cell line, e.g., a THP1, Raw264.7, IC21, P388D1, U937, or HL60 cell line.
In another example, an iRNA-lipophilic conjugate can be delivered to lymphoma, or leukemia cell line, e.g., an SEM-K2, WEHI-231, HB56, TIB55, Jurkat, K562, EL4, LRMB, Bcl-1, or TF1 cell line. For example, an iRNA-lipophilic conjugate can be delivered to a lymphoma cell line to target a specific gene for down regulation. An iRNA-lipophilic agent can target (down-regulate) a gene in a Jurkat cell line, for example, that encodes an immune factor, such as an interleukin gene, e.g., IL-1, IL-2, IL-5, IL-6, IL-8, IL-10, IL-15, IL-16, IL-17, or IL-18. In another aspect, an iRNA-lipophilic conjugate can target a gene that encodes a receptor of an interleukin.
An iRNA-lipophilic conjugate can target a gene resulting from a chromosomal translocation, such as BCR-ABL, TEL-AML-1, EWS-FLI1, EWS-ERG, TLS-FUS, PAX3-FKHR, or AML1-ETO. For example, an iRNA-lipophilic conjugate that targets a gene resulting from a chromosomal translocation can be delivered to a leukemia cell line, e.g., any of the leukemia cell lines discussed above.
An iRNA-lipophilic conjugate can be delivered to an immortalized cell line, including immortalized cell lines from a variety of different tissue types, including but not limited to T-cells, fibroblast cells, epithelial cells (e.g., kidney epithelial cells) and muscle cells (e.g., smooth muscle cells). Exemplary immortalized cell lines are CTLL-2 (T-cell), Rat 6 (fibroblast), VERO (fibroblast), MRC5 (fibroblast), CV1 (fibroblast), Cos7 (fibroblast), RPTE (kidney epithelial), and A10 (smooth muscle) cell lines.
An iRNA-lipophilic conjugate can be delivered to a mast cell line, for example. An iRNA-lipophilic conjugate delivered to a mast cell line can target, for example, a gene encoding a GRB2 associated binding protein (e.g., GAB2).
An iRNA-lipophilic conjugate can be delivered to an adherent tumor cell line, including tumor cell lines from a variety of different tissue types including but not limited to cancers of the bladder, lung, breast, cervix, colon, pancreas, prostate, and liver, melanomas, and glioblastomas.
Exemplary tumor cell lines include the T24 (bladder), J82 (bladder), A549 (lung), Calu1 (lung), SW480 (colon), SW620 (colon), CaCo2 (colon), A375 (melanoma), C8161 (melanoma), MCF-7 (breast), MDA-MB-231 (breast), HeLa (cervical), HeLa S3 (cervical), MiaPaCall (pancreas), Panc1 (pancreas), PC-3 (prostate), LNCaP (prostate), HepG2 (hepatocellular), and U87 (glioblastoma) cell lines. An iRNA-lipophilic conjugate that targets a specific gene can be delivered to an adherent tumor cell line. For example, an iRNA-lipophilic conjugate that targets a growth factor or growth factor receptor, such as a TGF-beta (e.g., TGF-beta 1) or TGF-beta receptor gene, can be delivered to an A549 or HepG2 cell line, a DLD2 colon carcinoma line, or a SKOV3 adenocarcinoma cell line. Other exemplary target growth factor genes include platelet derived growth factor (PDGF) and PDGF-Receptor (PDGFR), vascular endothelial growth factor (VEGF) and VEGF receptor genes (e.g., VEGFr1, VEGFr2, or VEGFr3), and insulin-growth factor receptors, such as type I insulin-growth factor (IGF) receptors, including IGF-1R, DAF-2 and InR.
In another example, an iRNA-lipophilic conjugate that targets one or more genes in a protein tyrosine phosphatase type IVA (PRL3, also called PTP4A3) gene family (e.g., PRL1, PRL2, or PRL3), or a gene in a PRL3 pathway, can be delivered to an A549 cell line, or to a cultured colorectal epithelial cell line.
In another example, an iRNA-lipophilic conjugate can target one or more protein kinase C genes in an adherent tumor cell line, such as in a mouse Lewis lung carcinoma, B16 melanoma, mouse mammary adenocarcinoma or fibrosarcoma; or a human lung carcinoma, bladder carcinoma, pancreatic cancer, gastric cancer, breast cancer, thyroid carcinoma, or melanoma. An iRNA-lipophilic conjugate can target a gene encoding a PKC isoforms, such as PKC-alpha, PKC beta I, PKC beta II, PKC gamma, PKC delta, PKC epsilon, and/or PKC zeta, or a gene encoding one or more receptors of a protein kinase C polypeptide.
In another example, an iRNA-lipophilic conjugate can target a gene encoding a P-glycoprotein, such as a gene in the multidrug resistance (MDR) gene family, e.g., MDR1. An iRNA-lipophilic conjugate that targets an MDR gene can be delivered, for example, to a human KB carcinoma cell line, a human leukemia or ovarian carcinoma cell line, or a lung carcinoma cell line such as A549.
In another example, an iRNA-lipophilic conjugate can target a gene encoding a gene in the telomerase pathway, such as TERT or the telomerase template RNA (TR/TERC). An iRNA-lipophilic conjugate that targets a gene in the telomerase pathway can be delivered, for example, to a human cancer cell line, e.g., a breast, cervical, endometrial, meningeal, lung, testicular, or ovarian cancer cell line.
In another example, an iRNA-lipophilic conjugate delivered to an adherent cell line (e.g., a HeLa, parathyroid adenoma, or A549 cell line) can target a cyclin gene, such as cyclin D1.
In another example, an iRNA-lipophilic conjugate delivered to an adherent cell line (e.g., a HeLa cell line) can target an NF-kappaB or REL-A gene, or a gene encoding a ligand or receptor of an NF-kappaB or REL-A polypeptide, or a gene encoding a subunit of NF-kappaB, such as REL-B, REL, NF-kappaB 1 or NF-kappaB2.
In another example, an iRNA-lipophilic conjugate delivered to an adherent cell line (e.g., a HeLa or A549 cell line) can target a gene encoding proliferating cell nuclear antigen (PCNA), a checkpoint kinase gene (CHK-1), or a c-fos gene. Further, an iRNA-lipophilic conjugate can target any gene in a PCNA, CHK-1, or c-fos pathway. For example an iRNA-lipophilic conjugate can down-regulate a gene encoding jun, which is in the c-fos pathway.
In another example, an iRNA-lipophilic conjugate delivered to an adherent cell line (e.g., an A549, T24, or A375 cell line) can target a gene encoding BCL2.
The cell lines described herein can be used to test iRNA-lipophilic conjugates that target exogenous, such as pathogenic or viral, nucleic acids. For example, an iRNA-lipophilic conjugate that targets a hepatitis viral gene can be delivered to a human hepatoma cell line, such as a HepG2 or Huh cell line, e.g., Huh1, Huh4, Huh7, and the like, that has been infected with the virus (e.g., an HAV, HBV, or HCV). For example, an iRNA-lipophilic conjugate that targets an HCV gene, such as in an infected Huh cell line, can target a conserved region of the HCV genome, such as the 5′-non-coding region (NCR), the 5′ end of the core protein coding region, or the 3′-NCR.
The cell lines described herein can be also be used to test iRNA-lipophilic conjugates that target exogenous recombinant nucleic acids, such as reporter genes (e.g., GFP, lacZ, beta-galactosidase, and the like), that are transfected (transiently or stably) into the cell lines.
In one aspect, an iRNA-lipophilic conjugate can be delivered to a B-cell line, e.g., BC-3, C1R, or ARH-77 cells. In another aspect, an iRNA-lipophilic conjugate can be delivered to T-cells, e.g., J45.01, MOLT, and CCRF-CEM cells. An iRNA-lipophilic conjugate can target an endogenous or exogenous nucleic acid. For example, development of an iRNA-lipophilic conjugate that targets an HIV gene can be tested against an exogenous HIV nucleic acid in a B cell or T cell line, or in a macrophage or endothelial cell culture system.
An iRNA-lipophilic conjugate can be delivered to cells derived from endoderm, epithelium, or mesoderm. For example, an iRNA-lipophilic conjugate can be delivered to cells of the HeLa or MCF7 epithelial cell lines, to cells of the HUVEC endothelial cell line, or to cells of an SK-UT or HASMC mesodermal cell line. In one example, an iRNA-lipophilic agent that targets a TGF-beta nucleic acid or TGF-beta receptor nucleic acid can be delivered to a vascular smooth muscle cell line, e.g., the kidney fibroblast 293 cell line. Other exemplary targets of iRNA-lipophilic conjugates delivered to fibroblast cells, such as 293 cells, included a protein tyrosine phosphatase-1B (PTP-1B) gene or MAP kinase gene (e.g., ERK1, ERK2, JNK1, JNK2, and p38). In another example, an iRNA-lipophilic conjugate that targets an MDR gene for down-regulation can be delivered to the human intestinal epithelial cell line, Caco-2.
In one example, an iRNA-lipophilic conjugate delivered to a cell line, such as an epithelial or mesodermal cell line (e.g., a HeLa or HASMC cell line, respectively), can target a gene encoding a Myc or Myb polypeptide, e.g., c-Myc, N-Myc, L-Myc, c-Myb, a-Myb, b-Myb, and v-Myb, or a gene in the Myc or Myb gene pathway, such as cyclin D1, cyclin D2, cyclin E, CDK4, cdc25A, CDK2, or CDK4.
In one example, an iRNA-lipophilic conjugate that targets a gene expressed in the nervous system, such as in the brain, e.g, a G72 or D-amino acid oxidase (DAAO) gene, can be delivered to a cultured neuronal cell line, such as an hNT cell line.
In another example, an iRNA-lipophilic conjugate can target a gene encoding a gene in the telomerase pathway, such as TERT or TR/TERC. An iRNA-lipophilic conjugate that targets a gene in the telomerase pathway can be delivered, for example, to a human keratinocyte cell line, such as a HEK cell line, e.g., HEKn or HEKa.
In another example, an iRNA-lipophilic conjugate delivered to a tissue-specific cell-line, such as a HEK (keratinocyte), HuVEC (endothelial), 3T3 (fibroblast), or NHDF (fibroblast) cell line, can target a gene encoding BCL-2, or VEGF or a VEGF receptor (e.g., VEGFr1, VEGFr2, or VEGFr3).
An iRNA-lipophilic conjugate can be delivered to a subgroup of cells derived from a particular tissue. For example, an iRNA-lipophilic conjugate can be delivered to a proximal tubular kidney cell line, such as the mouse cell line mIMCD-3. An iRNA-lipophilic conjugate that targets a TGF-beta nucleic acid or TGF-beta receptor nucleic acid, for example, can be delivered to a cell line derived from prostate tissue, e.g., a PC3 or RWPE prostate cell line. An iRNA-lipophilic conjugate delivered to a prostate tissue cell line can alternatively target a polycomb group gene, such as EZH2.
In another example, an iRNA-lipophilic conjugate can be delivered to pancreatic islet b-cells, where for example, it targets a gastric inhibitory polypeptide (GIP) gene, or a GIP-receptor gene.
The iRNA-lipophilic conjugates described herein are not limited in the cell lines to which they can be applied or to the nucleic acids to which they can target.
iRNA Agent Structure
The monomers described herein can be used to make oligonucleotides which are useful as iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi, e.g., with respect to an endogenous gene of a subject or to a gene of a pathogen. In most cases the iRNA agent will incorporate monomers described herein together with naturally occurring nucleosides or nucleotides or with other modified nucleosides or nucleotides. The modified monomers can be present at any position in the iRNA agent, e.g., at the terminii or in the middle region of an iRNA agent or in a duplex region or in an unpaired region. In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, a hairpin or other single strand structure or a two-strand structure, as described herein.
An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.
The RRMS-containing iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.
As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the ribose replacement modification subunit (RRMS). Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.
iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.
Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2-3 nucleotides.
In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:
(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below;
(2) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group;
(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an antisense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;
(4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C3′-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C3′-endo pucker in all, or at least 50, 75, 80, 85, 90, or 95% of its sugars; exhibit a C3′-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar which is not a C3′-endo pucker structure. These limitations are particularly preferably in the antisense strand;
(5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.
A “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). (These modifications can also be used with the antisense strand of a double stranded iRNA.) A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50 nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.
Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.
A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.
The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.
The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.
In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents
It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykänen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5′-end. Antisense strand modifications include 5′ phosphorylation as well as any of the other 5′ modifications discussed herein, particularly the 5′ modifications discussed above in the section on single stranded iRNA molecules.
It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.
Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.
The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides.
As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.
In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” (excluding the RRMS containing subunit(s)) to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.
As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.
RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.
Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.
As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.
In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.
Modifications and nucleotide surrogates are discussed below.
The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.
Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Unmodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.
Modified nucleic acids and nucleotide surrogates can include one or more of:
(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5′ end of a nucleic acid and the terminal Z position at the 3′ end of a nucleic acid, are within the term “linking phosphate oxygens” as used herein.);
(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;
(iii) wholesale replacement of the phosphate moiety (bracket I) with “dephospho” linkers;
(iv) modification or replacement of a naturally occurring base;
(v) replacement or modification of the ribose-phosphate backbone (bracket II);
(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.
The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.
It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X═O and Y═N in the above figure.
Specific modifications are discussed in more detail below.
The Phosphate Group
The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.
Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.
The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3′) or position Z (5′). Replacement of W with carbon or Z with nitrogen is preferred.
Candidate agents can be evaluated for suitability as described below.
The Sugar Group
A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2′ alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2′ position is not possible.
Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH2CH2OCH3, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.
“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.
The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.
Modified RNAs can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.
To maximize nuclease resistance, the 2′ modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called “chimeric” oligonucleotides are those that contain two or more different modifications.
The modification can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.
Candidate modifications can be evaluated as described below.
Replacement of the Phosphate Group
The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.
Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonyl amino and methylenemethylimino groups.
Candidate modifications can be evaluated as described below.
Replacement of Ribophosphate Backbone
Oligonucleotide-mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.
Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.
Candidate modifications can be evaluated as described below.
Terminal Modifications
The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end, 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH2)n—, —(CH2)nN—, —(CH2)nO—, —(CH2)nS—, O(CH2CH2O)nCH2CH2OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′ end can be an —OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).
Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).
Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety; modifications useful for this include mitomycin C.
Candidate modifications can be evaluated as described below.
The Bases
Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications. Alternatively, substituted or modified analogs of any of the above bases, e.g., “unusual bases” and “universal bases” described herein, can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methyl aminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.
Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.
Candidate modifications can be evaluated as described below.
Evaluation of Candidate RNA's
One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.
A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.
In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.
References
General References
The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.
Modification described in WO 00/44895, WO01/75164, or WO02/44321 can be used herein.
The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.
Phosphate Group References
The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).
Replacement of the Phosphate Group References
Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.
Terminal Modification References
Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.
Bases References
N-2 substituted purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.
Preferred iRNA Agents
Preferred RNA agents have the following structure (see Formula 2 below):
Referring to Formula 2 above, R1, R2, and R3 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methyl aminomethyl-2-thiouracil, 3-(3-amino-3 carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
R4, R5, and R6 are each, independently, OR8, O(CH2CH2O)mCH2CH2OR8; O(CH2)nR9; O(CH2)nOR9, H; halo; NH2; NHR8; N(R8)2; NH(CH2CH2NH)mCH2CH2NHR9; NHC(O)R8; cyano; mercapto, SR8; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R4, R5, or R6 together combine with R7 to form an [—O—CH2—] covalently bound bridge between the sugar 2′ and 4′ carbons.
A1 is:
H; OH; OCH3; W1; an abasic nucleotide; or absent;
(a preferred A1, especially with regard to anti-sense strands, is chosen from 5′-monophosphate ((HO)2(O)P—O-5′), 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2—), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2—), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-)).
A2 is:
A3 is:
and
A4 is:
H; Z4; an inverted nucleotide; an abasic nucleotide; or absent.
W1 is OH, (CH2)nR10, (CH2)NHR10, (CH2)n OR10, (CH2)n SR10; O(CH2)nR10;
O(CH2)nOR10, O(CH2)nNR10, O(CH2)nSR10; O(CH2)nSS(CH2)nOR10, O(CH2)nC(O)OR10, NH(CH2)nR10; NH(CH2)nNR10; NH(CH2)nOR10, NH(CH2)nSR10; S(CH2)nR10, S(CH2)nNR10, S(CH2)nOR10, S(CH2)nSR10O(CH2CH2O)mCH2CH2OR10; O(CH2CH2O)mCH2CH2NHR10, NH(CH2CH2NH)mCH2CH2NHR10; Q-R10, O-Q-R10N-Q-R10, S-Q-R10 or —O—. W4 is O, CH2, NH, or S.
X1, X2, X3, and X4 are each, independently, O or S.
Y1, Y2, Y3, and Y4 are each, independently, OH, O−, OR8, S, Se, BH3, H, NHR9, N(R9)2 alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.
Z1, Z2, and Z3 are each independently O, CH2, NH, or S. Z4 is OH, (CH2)nR1, (CH2)nNHR10, (CH2)n OR10, (CH2)n SR10; O(CH2)nR10; O(CH2)nOR10, O(CH2)nNR10, O(CH2)nSR10, O(CH2)nSS(CH2)nOR10, O(CH2)nC(O)OR10; NH(CH2)nR10; NH(CH2)nNR1; NH(CH2)nOR10, NH(CH2)nSR10; S(CH2)nR10, S(CH2)nNR10, S(CH2)nOR10, S(CH2)nSR10O(CH2CH2O)mCH2CH2OR10, O(CH2CH2O)mCH2CH2NHR10, NH(CH2CH2NH)mCH2CH2NHR10; Q-R10, O-Q-R10N-Q-R10, S-Q-R10.
x is 5-100, chosen to comply with a length for an RNA agent described herein.
R7 is H; or is together combined with R4, R5, or R6 to form an [—O—CH2—] covalently bound bridge between the sugar 2′ and 4′ carbons.
R8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R9 is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R10 is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.
Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):
Referring to Formula 3, A10-A40 is L-G-L; A10 and/or A40 may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH2(CH2)g; N(CH2)g; O(CH2)g; S(CH2)g. G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.
R10, R20, and R30 are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
R40, R5, and R60 are each, independently, OR8, O(CH2CH2O)mCH2CH2OR8; O(CH2)nR9; O(CH2)nOR9, H; halo; NH2; NHR8; N(R8)2; NH(CH2CH2NH)mCH2CH2R9; NHC(O)R8; cyano; mercapto, SR7; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R40, R5, or R60 together combine with R70 to form an [—O—CH2—] covalently bound bridge between the sugar 2′ and 4′ carbons.
x is 5-100 or chosen to comply with a length for an RNA agent described herein.
R70 is H; or is together combined with R40, R5, or R60 to form an [—O—CH2—] covalently bound bridge between the sugar 2′ and 4′ carbons.
R8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R9 is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.
Preferred nucleoside surrogates have the following structure (see Formula 4 below):
SLR100-(M-SLR200)x-M-SLR300 FORMULA 4
S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH2(CH2)g; N(CH2)g; O(CH2)g; S(CH2)g; —C(O)(CH2)n—Or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.
R100, R200, and R300 are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1, 2, 4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.
x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.
Nuclease Resistant Monomers
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, that incorporates a nuclease resistant monomer (NRM), such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/469,612, filed on May 9, 2003, and International Application No. PCT/US04/07070, both of which are hereby incorporated by reference.
An iRNA agent can include monomers which have been modified so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.
While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the half-life of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3′ and/or 5′ end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.
Modifications described herein can be incorporated into any double-stranded RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3′ end and/or the 5′ end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.
Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:
(i) chiral (Sp) thioates. Thus, preferred NRMs include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr2, or Br3. When X is S, enriched or chirally pure Sp linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRMs are discussed in more detail below;
(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRMs include monomers at the terminal position derivatized at a cationic group. As the 5′ end of an antisense sequence should have a terminal —OH or phosphate group this NRM is preferably not used at the 5′ end of an anti-sense sequence. The group should be attached at a position on the base which minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g, at the 5′ position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;
(iii) nonphosphate linkages at the termini. Thus, preferred NRMs include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3′ CH2-NCH3—O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′.;
(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus, preferred NRM's can included these structures;
(v) L-RNA, 2′-5′ linkages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphate and modified phosphate linkages (e.g., thiophosphates, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages; monomers having an alpha linkage at the 1′ site on the sugar, e.g., the structures described herein having an alpha linkage;
(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;
(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and
(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5′ position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an unprotected phosphate.
One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintained.
In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.
Chiral SP Thioates
A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moieties, SB1 and SB2.
In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein SP). Thus if 60% of a population of stereogenic phosphorus atoms have the Rp configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the SP configuration.
In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the SP configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the Rp configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the SP configuration and may be substantially free of stereogenic phosphorus atoms having the Rp configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the Rp configuration and may be substantially free of stereogenic phosphorus atoms having the SP configuration. As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the Rp configuration” means that moieties containing stereogenic phosphorus atoms having the Rp configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the SP configuration” means that moieties containing stereogenic phosphorus atoms having the SP configuration cannot be detected by conventional methods known in the art (chiral HPLC, 1H NMR analysis using chiral shift reagents, etc.).
In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the SP configuration and be substantially free of stereogenic phosphorus atoms having the Rp configuration.
Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate
at the 3′ end of a strand, while the latter can be used to introduce this modification at the 5′ end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R2′ can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH3), DMT is dimethoxytrityl, and “BASE” can be a natural, unusual, or a universal base.
X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the Rp configuration (i.e., being substantially free of the SP configuration) or only the SP configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the Rp configuration and about 50% of the atoms have the SP configuration. Dimers having stereogenic phosphorus atoms with the Rp configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the SP configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.
Cationic Groups
Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2′ position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).
Nonphosphate Linkages
Modifications can also include the incorporation of nonphosphate linkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.
3′-Bridging Thiophosphates and 5′-Bridging Thiophosphates; Locked-RNA, 2′-5′ Linkages, Inverted Linkages, α-Nucleosides; Conjugate Groups; Abasic Linkages; and 5′-Phosphonates and 5′-Phosphate Prodrugs
Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.
Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2′ position of a first sugar and the 5′ position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are each linked through the respective 3′ positions. Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the ca-configuration. Modifications can also include L-RNA.
Modifications can also include 5′-phosphonates, e.g., P(O)(O)2—X—C5′-sugar (X═CH2, CF2, CHF and 5′-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R]2CH2C5′-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular SN2 displacement can depart as episulfide to afford the underivatized phosphate group.
Modification can also include the addition of conjugating groups described elsewhere herein, which are preferably attached to an iRNA agent through any amino group available for conjugation.
Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it can minimize off-target silencing.
In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent—it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.
In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucleotides of the cleave site, in either direction.)
Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.
An iRNA agent can have a first and a second strand chosen from the following:
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;
a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;
a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;
a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end; and
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;
a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).
An iRNA agent can also target two sequences and can have a first and second strand chosen from:
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);
a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;
a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a first strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand) and
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);
a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;
a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;
a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).
Ribose Mimics
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, that incorporates a ribose mimic, such as those described herein and those described in copending co-owned U.S. Provisional Application Ser. No. 60/454,962, filed on Mar. 13, 2003, and International Application No. PCT/US04/07070, both of which are hereby incorporated by reference.
Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3′ ribose hydroxyl group, thereby causing it to be less susceptible to degradation.
The secondary hydroxyl group refers to an “OH” radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.
Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):
Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to “W” of the phosphate group. These spacers or linkers can include e.g., —(CH2)n—, —(CH2)nN—, —(CH2)nO—, —(CH2)nS—, O(CH2CH2O)nCH2CH2OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.
The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH3—, C1-C6 alkyl, C6-C10 aryl, H, O, O, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.
R1 and R3 are each, independently, hydrogen; or C1-C10 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.
R2 is hydrogen; C1-C10 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R2 may be taken together with with R4 or R6 to form a ring of 5-12 atoms.
R4 is hydrogen; C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R4 may be taken together with with R2 or R5 to form a ring of 5-12 atoms.
R5 is hydrogen, C1-C10 alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R5 may be taken together with with R4 to form a ring of 5-12 atoms.
R6 is hydrogen, C1-C100 alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R6 may be taken together with with R2 to form a ring of 6-10 atoms;
R7 is hydrogen, C1-C10 alkyl, or C(O)(CH2)qC(O)NHR9; T is hydrogen or a functional group; n and q are each independently 1-100; R8 is C1-C10 alkyl or C6-C10 aryl; and R9 is hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.
Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.
In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3′ or 5′ ribose sugar carbon of the RNA agent.
In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.
In still yet another subset of RNAi agent delivery modules, n is 1, and R2 and R6 are taken together to form a ring containing six atoms and R4 and R5 are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):
The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.
As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
Palindromes
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having a palindrome structure as described herein and those described in one or more of U.S. Provisional Application Ser. No. 60/452,682, filed Mar. 7, 2003; U.S. Provisional Application Ser. No. 60/462,894, filed Apr. 14, 2003; and International Application No. PCT/US04/07070, filed Mar. 8, 2004, all of which are hereby incorporated by reference. The iRNA agents of the invention can target more than one RNA region. For example, an iRNA agent can include a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. The first and second sequences of the iRNA agent can be on different RNA strands, and the mismatch between the first and second sequences can be less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%. The first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent can be in bimolecular form. The first and second sequences of the iRNA agent can be fully complementary to each other.
The first target RNA region can be encoded by a first gene and the second target RNA region can encoded by a second gene, or the first and second target RNA regions can be different regions of an RNA from a single gene. The first and second sequences can differ by at least 1 nucleotide.
The first and second target RNA regions can be on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example. The first target RNA region can include a nucleotide substitution, insertion, or deletion relative to the second target RNA region, or the second target RNA region can a mutant or variant of the first target region.
The first and second target RNA regions can comprise viral or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In addition, the first and second target RNA regions can correspond to hot-spots for genetic variation.
The compositions of the invention can include mixtures of iRNA agent molecules. For example, one iRNA agent can contain a first sequence and a second sequence sufficiently complementary to each other to hybridize, and in addition the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture can also include at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence can be sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. The first and second sequences can be on the same or different RNA strands, and the third and fourth sequences can be on the same or different RNA strands.
The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. The target RNA regions can correspond to genetic hot-spots.
Methods of making an iRNA agent composition can include obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region can be obtained or provided, where each RNA target corresponds to a different variant or mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent can be constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S), and the first and second sequences can be sufficiently complementary to hybridize.
Sequence analysis, e.g., to identify common mutants in the target gene, can be used to identify a region of the target gene that has high variability or mutational frequency. A region of the target gene having high variability or mutational frequency can be identified by obtaining or providing genotype information about the target gene from a population.
Expression of a target gene can be modulated, e.g., downregulated or silenced, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region.
An iRNA agent can include a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region. The first and second variant RNA target regions can correspond to first and second variants or mutants of a target gene, e.g., viral gene, tumor suppressor or oncogene. The first and second variant target RNA regions can include allelic variants, mutations (e.g., point mutations), or polymorphisms of the target gene. The first and second variant RNA target regions can correspond to genetic hot-spots.
A plurality of iRNA agents (e.g., a panel or bank) can be provided.
Other than Canonical Watson-Crick Duplex Structures
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand, such as those described herein and those described in U.S. Provisional Application Ser. No. 60/465,665, filed Apr. 25, 2003, and International Application No. PCT/US04/07070, filed Mar. 8, 2004, both of which are hereby incorporated by reference.
The use of “other than canonical Watson-Crick pairing” between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).
In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence).
Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can e, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.
Thus, in another aspect, the invention features, an iRNA agent which includes: a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.
Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA duplex.
The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.
The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick paring.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.
By way of example:
The monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
A non-naturally occurring or modified monomer or monomers can be chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.
An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.
When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 12.
The pair shown in FIG. 12 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
iRNA agents of the invention can include:
A sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:
The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner).
The monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;
Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.
The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.
The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer is one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick paring.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.
By way of example:
The monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.
A non-naturally occurring or modified monomer or monomers can be chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.
An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.
When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.
The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
iRNA agents of the invention can include:
A sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:
The monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);
The monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;
Optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.
The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.
The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.
A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.
The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.
In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.
E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.
In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.
By way of example:
the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;
the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G;
the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U; or
the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.
In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.
An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.
The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.
In another aspect, the invention features, an iRNA agent which includes:
a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differ in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.
In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.
An embodiment is illustrated in the FIGS. 13 and 14.
In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained position in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a target sequence. The determinations can be compared. An iRNA agent thus analysed can be used in the development of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or design optimized iRNA agents.
In another aspect, the invention features, a kit which includes one or more of the following an iRNA described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.
In another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.
iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.
The term “other than canonical Watson-Crick pairing” as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or (3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.
The term “off-target” as used herein, refers to as a sequence other than the sequence to be silenced.
Universal Bases: “wild-cards”; shape-based complementarity
Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by
‘inverse’ sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)
(Importance of terminal base pair hydrogen-bonding in 3′-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)
(Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)
(Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)
(Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656)
(Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)
(1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)
(Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)
(1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self-pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)
(A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)
(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal complexes. Weizman, H.; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)
(Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)
(Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209)
(Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)
(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside. J. Org. Chem., 2001, 66, 2067-2071)
(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K.; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K.; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001, 11, 335-337)
Asymmetrical Modifications
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, that can be asymmetrically modified as described herein, and as described in International Application Serial No. PCT/US04/07070, filed Mar. 8, 2004, which is hereby incorporated by reference.
An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.
While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or “asymmetrical” functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand. In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2′ modification, e.g., 2′ OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5′ or 3′ end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2′OMe is present in the sense strand, and a targeting moiety is added to either the 5′ or 3′ end of the sense strand of the iRNA agent.
In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.
Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.
In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.
In an embodiment one strand has an asymmetrical 2′ modification, e.g., a 2′ OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2′ OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2′-O alkyl, preferably, 2′-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2′-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3′ overhangs of about 2 subunits in length.
Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2′, 6′, 7′, or 8′ position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.
Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.
Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids, 2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.
iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5′ end of the sense strand and the 3′end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5′ end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5′ exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a “tight” structure at this end of the molecule.
The end region of the iRNA agent defined by the 3′ end of the sense strand and the 5′end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an “open” structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.
Exemplary modifications for asymmetrical placement in the sense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;
(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;
(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;
(h) conjugate groups (preferably at terminal positions), e.g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.
Exemplary modifications for asymmetrical placement in the antisense strand include the following:
(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S);
(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);
(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the 3′ end); e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with RISC enzyme activity such as kinase activity;
(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with kinase activity;
(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5′ end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a “loose” structure in the 5′ region, additionally, they may interfere with kinase activity;
(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);
(g) cationic groups and Zwitterionic groups (preferably at a terminus);
conjugate groups (preferably at terminal positions), e.g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.
The 5′-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3′ end, particularly in the 3′ overhang.
In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.
The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50% activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.
The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.
In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.
Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.
Table 2 shows examples having strand I with a selected modification and strand II with a selected modification.
TABLE 2
Exemplary strand I- and strand II-modifications
Strand I
Strand II
Nuclease Resistance (e.g., 2′-OMe)
Biodistribution (e.g., P═S)
Biodistribution conjugate
Protein Binding Functionality
(e.g., Lipophile)
(e.g., Naproxen)
Tissue Distribution Functionality
Cell Targeting Functionality
(e.g., Carbohydrates)
(e.g., Folate for cancer cells)
Tissue Distribution Functionality
Fusogenic Functionality
(e.g., Kidney Cell Targetingmoieties)
(e.g., Polyethylene imines)
Cancer Cell Targeting
Fusogenic Functionality
(e.g., RGD peptides and imines)
(e.g., peptides)
Nuclease Resistance (e.g., 2′-OMe)
Increase in binding Affinity
(5-Me—C, 5-Me—U, 2-thio-U,
2-amino-A, G-clamp, LNA)
Tissue Distribution Functionality
RISC activity improving
Functionality
Helical conformation changing
Tissue Distribution Functionality
Functionalities
(P═S; lipophile, carbohydrates)
Z—X-Y Architecture
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent, having a Z—X-Y architecture or structure such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/510,246, filed on Oct. 9, 2003, which is hereby incorporated by reference, copending, co-owned U.S. Provisional Application Ser. No. 60/510,318, filed on Oct. 10, 2003, which is hereby incorporated by reference, and copending, co-owned International Application No. PCT/US04/07070, filed Mar. 8, 2004.
Thus, an iRNA agent can have a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:
Z—X—Y.
It may be desirable to modify subunits in one or both of Z and/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.
The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3′ or 5′ terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3-overhang. Typical inter-subunit bonds in the Z region include: P═O; P═S; S—P═S; P—NR2; and P—BR2. Chiral P═X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3′-OR, 3′SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alpha configuration bases; and 2′ arabino modifications.
The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X's will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2′-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2′OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2′OH subunits.
The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and infact, one will usually be a 3′ overhang and one will usually be a 5′ overhang.
In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2′-OH remains unsubstituted.
The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occurrence is minimized and in particular it is preferred that consecutive modified linkages be avoided.
In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2′-OH remains unsubstituted.
As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.
Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particularly preferred iRNA agents will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particularly preferred X regions will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.
In a preferred embodiment Y and/or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.
In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2′ moieties.
In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.
In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.
In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
In a preferred embodiment each of Y and/or Z independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.
In a preferred embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2′ moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.
A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Y and/or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.
An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some embodiments it is preferred that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.
An RRMS (described elsewhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.
Differential Modification of Terminal Duplex Stability
In one aspect, the monomers and methods described herein can be used to prepare an iRNA agent having differential modification of terminal duplex stability (DMTDS).
In addition, the monomers and methods described herein can be used to prepare iRNA agents having DMTDS and another element described herein. E.g., the monomers and methods described herein can be used to prepare an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the kidney, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.
iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5′ end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5′end. While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5′ end of the antisense strand.
The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3′ end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5′end.
Modifications which increase the tendency of the 5′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3′ end of the duplex to dissociate. Likewise, modifications which decrease the tendency of the 3′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5′ end of the duplex to dissociate.
Decreasing the Stability of the AS 5′ End of the Duplex
Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:
A:U is preferred over G:C;
G:U is preferred over G:C;
I:C is preferred over G:C (I=inosine);
mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;
pairings which include a universal base are preferred over canonical pairings.
A typical ds iRNA agent can be diagrammed as follows:
S
5′
R1
N1
N2
N3
N4
N5
[N]
N−5
N−4
N−3
N−2
N−1
R2 3′
AS
3′
R3
N1
N2
N3
N4
N5
[N]
N−5
N−4
N−3
N−2
N−1
R4 5′
S:AS
P1
P2
P3
P4
P5
[N]
P−5
P−4
P−3
P−2
P−1
5′
S indicates the sense strand; AS indicates antisense strand; R1 indicates an optional (and nonpreferred) 5′ sense strand overhang; R2 indicates an optional (though preferred) 3′ sense overhang; R3 indicates an optional (though preferred) 3′ antisense sense overhang; R4 indicates an optional (and nonpreferred) 5′ antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and Px, indicates a paring of sense Nx and antisense Nx. Overhangs are not shown in the P diagram. In some embodiments a 3′ AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3'S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)
It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5′ end of the AS strand. The terminal pair (the most 5′ pair in terms of the AS strand) is designated as P−1, and the subsequent pairing positions (going in the 3′ direction in terms of the AS strand) in the duplex are designated, P−2, P−3, P−4, P−5, and so on. The preferred region in which to modify to modulate duplex formation is at P−5 through P−1, more preferably P−4 through P−1, more preferably P−3 through P−1. Modification at P−1, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:
A:U
G:U
I:C
mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.
In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are pairs which promote dissociation.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are A:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are G:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are I:C.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are pairings which include a universal base.
Increasing the Stability of the AS 3′ End of the Duplex
Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:
G:C is preferred over A:U
Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings
analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C)
2-amino-A:U is preferred over A:U 2-thio U or 5 Me-thio-U:A are preferred over U:A
G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G
guanadinium-G-clamp:G is preferred over C:G
pseudo uridine:A is preferred over U:A
sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3′ end of the AS strand. The terminal pair (the most 3′ pair in terms of the AS strand) is designated as P1, and the subsequent pairing positions (going in the 5′ direction in terms of the AS strand) in the duplex are designated, P2, P3, P4, P5, and so on. The preferred region in which to modify to modulate duplex formation is at P5 through P1, more preferably P4 through P1, more preferably P3 through P1. Modification at P1, is particularly preferred, alone or with modification(s) at other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:
G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)
2-amino-A:U
2-thio U or 5 Me-thio-U:A
G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clamp: G
pseudo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.
In a preferred embodiment the at least 2, or 3, of the pairs in P−1, through P−4, are pairs which promote duplex stability.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are G:C.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are a pair having an analog that increases stability over Watson-Crick matches.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are 2-amino-A:U.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are 2-thio U or 5 Me-thio-U:A.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are G-clamp:G.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are guanidinium-G-clamp: G.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are pseudo uridine:A.
In a preferred embodiment the at least 2, or 3, of the pairs in P1, through P4, are a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhances binding.
G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., “Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., “Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nuclease resistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al., “A cytosine analog that confers enhanced potency to antisense oligonucleotides,” Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.
Simultaneously Decreasing the Stability of the AS 5′End of the Duplex and Increasing the Stability of the AS 3′ End of the Duplex
As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5′end of the duplex and increase the stability of the AS 3′ end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5′ end of the duplex with one or more of the stability increasing modifications in the AS 3′ end of the duplex. Accordingly a preferred embodiment includes modification in P−s through P−1, more preferably P−4 through P−1 and more preferably P−3 through P−1. Modification at P−1, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5′ end of the duplex region be chosen independently from the group of:
A:U
G:U
I:C
mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and
a modification in P5 through P1, more preferably P4 through P1 and more preferably P3 through P1. Modification at P1, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3′ end of the duplex region be chosen independently from the group of:
G:C
a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)
2-amino-A:U
2-thio U or 5 Me-thio-U:A
G-clamp (an analog of C having 4 hydrogen bonds):G
guanadinium-G-clamp: G
pseudo uridine:A
a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.
The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the
AS strand, to provide a desired level of DMTDS.
The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P−5 through P−1 and/or at least one P in P5 through P1 to provide a DMTDS iRNA agent.
DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or 3'S strand overhangs) which self associate to form intrastrand duplex structure.
Preferably these iRNA agents will have an architecture described herein.
Other Embodiments
An RNA, e.g., an iRNA agent, can be produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (U.S. Pat. No. 5,328,470), or by stereotactic injection (see, e.g., Chen et al., Proc. Natl. Acad. Sci. USA 91:3054-3057, 1994). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that includes the top strand of an iRNA agent and one that produces a transcript that includes the bottom strand of an iRNA agent. When the templates are transcribed, the iRNA agent is produced, and processed into sRNA agent fragments that mediate gene silencing.
In Vivo Delivery
An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.
The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems, as described in U.S. Pat. No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.
A composition for iRNA delivery can employ an “inclusion complex,” a molecular compound having the characteristic structure of an adduct. In this structure, the “host molecule” spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the “guest molecule”) is situated in the cavity of the host molecule without affecting the framework structure of the host. A “host” is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference.
Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of “host-guest” complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a kidney cell), intercellular trafficking, and/or cell entry and release.
Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.
Physiological Effects
The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.
The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.
Delivery Module
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent described herein, that can be used with a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar. 12, 2003, and International Application Serial No. PCT/US04/07070, filed Mar. 8, 2004, both of which are hereby incorporated by reference.
The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell.
An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the kidney.
The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.
The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):
(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),
(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and
(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.
Carrier Agents
The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable.
The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.
A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.
Fusogenic Agents
A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.
In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.
The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.
In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin.
A fusogenic agent can have one or more of the following characteristics: (a) is at least 1 Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.
Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.
Condensing Agent
The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.
A condensing agent can have the following characteristics: (a) at least 1 Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.
Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.
Amphipathic Delivery Agents
The monomers and methods described herein can be used to prepare an RNA, e.g., an iRNA agent described herein, that can be used with an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/455,050, filed on Mar. 13, 2003, and International Application Serial No. PCT/US04/07070, filed Mar. 8, 2004, which is hereby incorporated by reference.
An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid by layer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs described herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.
One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hydrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) “Protein design and folding: template trapping of self-assembled helical bundles” J Pept Sci 7(3):146-51; Chen et al. (2002) “Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix” J Pept Res 59(1):18-33; Iwata et al. (1994) “Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation” J Biol Chem 269(7):4928-33; Cornut et al. (1994) “The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin” FEBS Lett 349(1):29-33; Negrete et al. (1998) “Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices,” Protein Sci 7(6):1368-79.
Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.
The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.
An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.
The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.
In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.
The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.
In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):
The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.
In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.
The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.
The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.
The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.
In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):
Referring to Formula M, R1 is C1-C100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C1-C100 perfluoroalkyl; or OR5.
R2 is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR6; or C1-C100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO2, alkenyl, or alkynyl.
R3 is hydrogen, or when taken together with R4 froms a fused phenyl ring.
R4 is hydrogen, or when taken together with R3 froms a fused phenyl ring.
R5 is C1-C100 alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C1-C100 perfluoroalkyl; and R6 is C1-C100 alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO2, alkenyl, or alkynyl.
Increasing Cellular Uptake of dsRNAs
A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug can have a transient effect on the cell.
The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.
iRNA conjugates
An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.
iRNA Production
An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.
Organic Synthesis
An iRNA can be made by separately synthesizing each respective strand of a double-stranded RNA molecule. The component strands can then be annealed.
A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.
Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.
dsRNA Cleavage
iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used:
In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribe™ RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.
In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50. dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present.
Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.
Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.
Formulation
The iRNA agents described herein can be formulated for administration to a subject
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.
A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.
The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA composition is formulated in a manner that is compatible with the intended method of administration (see, below).
In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.
A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.
In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.
Exemplary formulations are discussed below:
Liposomes
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types, e.g., to cells of the kidney, such as those described herein.
A liposome containing a iRNA can be prepared by a variety of methods.
In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of iRNA.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.
Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes.
Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.
Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.
Surfactants
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant. In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Micelles and other Membranous Formulations
For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.
Particles
For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.
Sustained-Release Formulations.
An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time.
Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 μm rather than 10 μm. The time of mixing also can be altered.
Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.
Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.
Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a ⅛″ microtip probe, at setting #3.
Spray-Drying.
An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA.
Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.
Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.
Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.
Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.
Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.
The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By “associated with or comprise” it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.
Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.
Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C6-C22) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, disteroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidyl ethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.
Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic® F68), poloxamer 407 (Pluronic® F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.
In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP, 1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.
For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include “Parubisu [phonetic rendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”, manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray Drier L-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,” manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer.
While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50° C. to about 200° C., preferably between about 50° C. and 100° C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0° C. and about 150° C., preferably between 0° C. and 90° C., and even more preferably between 0° C. and 60° C.
The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 μm or less with about 90% of the mass being in particles having a diameter less than 5 μm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 μm with about 80% of the mass of the particles having a diameter of less than 5 μm.
The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.
Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 μm to 100 μm. A preferred carrier material is crystalline lactose having a size in the above-stated range.
Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers.
Methods for spray drying hydrophobic and other drugs and components are described in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Pharm. Acta Helv 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP 806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP 4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870.
Lyophilization.
An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.
Targeting
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.
In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen of the kidney (e.g., G250, CA15-3, CA19-9, CEA, or HER2/neu) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).
Table 3 provides a number of antigens which can be used to target an iRNA to a selected cell, such as when targeting of the iRNA agent to a tissue other than the kidney is desired.
TABLE 3
Targeting Antigens
ANTIGEN
Exemplary tumor tissue
CEA (carcinoembryonic antigen)
colon, breast, lung
PSA (prostate specific antigen)
prostate cancer
CA-125
ovarian cancer
CA 15-3
breast cancer
CA 19-9
breast cancer
HER2/neu
breast cancer
α-feto protein
testicular cancer, hepatic cancer
β-HCG (human chorionic
testicular cancer, choriocarcinoma
gonadotropin)
MUC-1
breast cancer
Estrogen receptor
breast cancer, uterine cancer
Progesterone receptor
breast cancer, uterine cancer
EGFr (epidermal growth
bladder cancer
factor receptor)
In one embodiment, the targeting moiety is attached to a liposome. For example, U.S. Pat. No. 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety. For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.
Genes and Diseases
In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation. The method includes:
providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;
administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject,
thereby treating the subject.
In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.
In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.
In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.
In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.
In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.
In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.
In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia.
In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JNK expression, e.g., pancreatic or breast cancers.
In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.
In a preferred embodiment the iRNA agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.
In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.
In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.
In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.
In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.
In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.
In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.
In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers.
In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.
In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.
In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers.
In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.
In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.
In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.
In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma.
In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.
In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.
In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.
In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.
In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.
In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.
In a preferred embodiment the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAF1/CIP1) expression, e.g., liver cancer.
In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.
In a preferred embodiment the iRNA agent silences mutations in the PPM1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1D expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.
In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.
In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).
In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.
In another preferred embodiment the iRNA agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.
In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.
In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.
In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma
In a preferred embodiment the iRNA agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma
In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.
In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.
In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.
In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.
In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.
In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TEL/AML1 fusion gene expression, e.g., childhood acute leukemia.
In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma.
In another preferred embodiment the iRNA agent silences the TLS/FUS 1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.
In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.
In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.
In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:
providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;
administering the iRNA agent to a subject,
thereby treating the subject.
In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin.
In a preferred embodiment the iRNA agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis.
In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.
In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.
In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection. The method includes:
providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;
administering the iRNA agent to a subject, preferably a human subject,
thereby treating the subject.
Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.
In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.
In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced.
The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).
In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCR5, Gag, or Rev.
In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD4 or Tsg101.
The invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.
In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.
In preferred embodiment the expression of a human gene that is required for HBV replication is reduced.
The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.
In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced.
The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis
In a preferred embodiment, the expression of a HCV gene is reduced.
In another preferred embodiment the expression of a human gene that is required for HCV replication is reduced.
The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.
In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.
In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.
Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.
In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.
In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced.
Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.
In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.
In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced.
The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.
In a preferred embodiment, the expression of a CMV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced.
Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.
In a preferred embodiment, the expression of a EBV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced.
Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.
In a preferred embodiment, the expression of a KSHV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced.
The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).
In a preferred embodiment, the expression of a JCV gene is reduced.
In preferred embodiment the expression of a human gene that is required for JCV replication is reduced.
Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.
In a preferred embodiment, the expression of a myxovirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.
Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.
In a preferred embodiment, the expression of a rhinovirus gene is reduced.
In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.
Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.
In a preferred embodiment, the expression of a coronavirus gene is reduced.
In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.
Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.
In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5.
In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced.
Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.
In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.
In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.
Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.
In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.
Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.
In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.
The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.
In a preferred embodiment, the expression of a dengue virus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.
Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.
In a preferred embodiment, the expression of a SV40 gene is reduced.
In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced.
The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.
In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV1 gene is the Tax transcriptional activator.
In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced.
Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.
In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.
Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.
In a preferred embodiment, the expression of a EMCV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced.
The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.
In a preferred embodiment, the expression of a MV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for MV replication is reduced.
The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).
In a preferred embodiment, the expression of a VZV gene is reduced.
In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced.
The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.
In a preferred embodiment, the expression of an adenovirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.
The invention includes a method for treating patients infected by a yellow fever virus (YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.
In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.
In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced.
Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.
In a preferred embodiment, the expression of a poliovirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced.
Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox
In a preferred embodiment, the expression of a poxvirus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.
In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes: providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA is homologous to and can silence, e.g., by cleavage of a pathogen gene;
administering the iRNA agent to a subject, prefereably a human subject,
thereby treating the subject.
The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.
Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.
In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA1).
In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.
In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.
In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.
The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.
In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.
In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.
In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.
In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection
In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.
The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection
In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.
In a preferred embodiment the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.
In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder. The method includes:
providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response;
administering the iRNA agent to a subject,
thereby treating the subject.
In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplantated organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.
In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.
In a preferred embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.
In a preferred embodiment the disease or disorder is inflammation associated with an infection or injury.
In a preferred embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.
In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.
In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1).
In particularly preferred embodiments the iRNA agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.
In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR3.
In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, 1-309.
In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes:
providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain;
administering the iRNA to a subject,
thereby treating the subject.
In particularly preferred embodiments the iRNA agent silences a component of an ion channel.
In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand.
In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes: providing an iRNA agent which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder;
administering the iRNA agent to a subject,
thereby treating the subject.
In a preferred embodiment the disease or disorder is Alzheimer's Disease or Parkinson Disease.
In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein.
In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCA7 or SCA8.
In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCA7, SCA8.
The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.
E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR2A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S 14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.
Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes:
optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell;
providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;
administering the iRNA to the subject,
thereby treating the disorder.
The invention also includes a iRNA agent disclosed herein, e.g, an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene
In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:28) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.
The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.
Route of Delivery
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.
Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches.
In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.
Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.
For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.
Topical Delivery
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. “Topical administration” refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.
The term “skin,” as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μm and 0.2 mm thick, depending on its location on the body.
Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.
One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.
The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.
Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.
Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.
In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.
The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.
Pulmonary Delivery
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.
Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.
The term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be “respirable.” Preferably the average particle size is less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0 m. Usually the particle size distribution is between about 0.1 m and about 5 m in diameter, particularly about 0.3 m to about 5 m.
The term “dry” means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.
The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.
The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.
The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.
The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.
Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin; and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.
Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.
Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.
Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.
Oral or Nasal Delivery
For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.
In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.
The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.
A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.
Devices
For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.
Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.
Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.
In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.
Tissue, e.g., cells or organs, such as the kidney, can be treated with An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.
The tissue can be autologous, allogeneic, or xenogeneic tissue. For example, tissue (e.g., kidney) can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue, such as in the kidney. In another example, tissue containing hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.
Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.
In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.
In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide.
Dosage
In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×1016 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA, such as an RNA present in the kidney. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.
In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.
In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent.
In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.
The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.
In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a kidney disease, e.g., early stage renal disease, can be administered an iRNA agent specific for a target gene known to enhance the progression of the disease in conjunction with a drug known to inhibit activity of the target gene product. For example, a patient who has early stage renal disease can be treated with an iRNA agent that targets an SGLT2 RNA, in conjunction with the small molecule phlorizin, which is known to block sodium-glucose cotransport and to subsequently reduce single nephron glomerular filtration rate. In another example, a patient being treated for a cancer of the kidney can be administered an iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy.
Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).
The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.
Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.
Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.
The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.
Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.
The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.
The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.
Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.
Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.
Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.
Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.
Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.
Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.
iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.
Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.
Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue
iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.
An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.
The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned above.
Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.
A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.
A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.
The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.
The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.
A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques
In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load).
In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.
In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.
In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.
In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.
In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-10 alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof.
In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.
In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.
In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof. In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.
In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.
In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.
In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.
In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles. In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.
In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.
In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.
In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.
In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.
In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, α-lactalbumin, trypsinogen, and polyalanine.
In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.
In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.
In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.
As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.
The invention is further illustrated by the following examples, which should not be construed as further limiting.
EXAMPLES
Example 1
Diethyl2-azabutane-1,4-dicarboxylate AA
A 4.7M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until the completion of reaction was ascertained by TLC (19 h). After 19 h which it was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
Example 2
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic Acid Ethyl Ester AB
Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl2-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. the completion of the reaction was ascertained by TLC. The reaction mixture was concentrated in vacuum and to the ethylacetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB
Example 3
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic Acid Ethyl Ester AC
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at O ° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated in vacuum and the residue water was added and the product was extracted with ethyl acetate. The crude product was purified by converting into hydrochloride salt.
Example 4
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic Acid Ethyl Ester AD
Hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken in dichloromethane. The suspension was cooled to 0° C. with ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified flash chromatography (10.3 g, 92%).
Example 5
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic Acid Ethyl Ester AE
Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. and 5 g (6.6 mmol) of diester was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted with two 100 mL of dichloromethane and the combined organic extracts were washed twice with 10 mL of phosphate buffer, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were converted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to a residue. The residue was purified by column chromatography using 25% ethylacetate/hexanes to afford 1.9 g of β-ketoester was obtained (39%).
Example 6
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic Acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester AF
Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated in vacuum to yield the product which purified by column chromatography (10% MeOH/CHCl3). (89%).
Example 7
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic Acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester
Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature for overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated in vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3). (1.75 g, 95%)
Example 8
Succinic Acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H Cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) Ester AH
Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Example 9
Cholesterol Derivatised CPG AI
Succinate AH (0.254 g, 0.242 mmol) was dissolved in mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 μm/g) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (37 μM/g).
Example 10
(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H Cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) Phosphoramidite AJ
Compound AG (0.15 g, 0.158 mmol) was coevaporated with toluene (5 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.0089 g, 0.079 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in the mixture of anhydrous acetonitrile/dichloromethane (2:1, 1 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (0.0714 g, 0.0781 mL, 0.237 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC (1:1 ethyl acetate:hexane). The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (10 mL) and washed with 5% NaHCO3 (4 mL) and brine (4 mL). The ethyl acetate layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting mixture was chromatographed (50:49:1, EtOAc:Hexane:triethlyamine) to afford AJ as white foam (0.152 g, 84%).
Example 11
RNA Synthesis, Deprotection and Purification Protocol
1. Synthesis:
The RNA molecules were synthesized on a 394 ABI machine using the standard 93 step cycle written by the manufacturer with modifications to a few wait steps as described below. The solid support was controlled pore glass (CPG, 1 mole, 500A, Glen Research, Sterling Va.) and the monomers were RNA phosphoramidites with standard protecting groups (N6-benzoyl-5′-O-dimethoxytrityladenosine-2′ tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityluridine-2′tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, N2-isobutyryl-5′-O-dimethoxytritylguanosine-2′tbutyldimethylsilyl, 3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and N4-benzoyl-5′-O-dimethoxytritylcytidine-2′tbutyldimethylsilyl-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite from Chemgenes Corp MA) used at a concentration of 0.15M in acetonitrile (CH3CN) and a coupling time of 7.5 min. The activator was thiotetrazole (0.25M), For the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation Beaucage reagent 0.5M solution in acetomitrile was used. All reagents for synthesis were also from Glen Research.
2. Deprotection-I (Oligomer Cleavage, Base and Phosphate Deprotection)
After completion of synthesis the controlled pore glass (CPG) was transferred to a screw cap vial (Fisher, catalog number 03-340-5N) or a screw cap RNase free microfuge tube. The oligonucleotide was cleaved from the CPG with simultaneous deprotection of base and phosphate groups with 1.0 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 6 hours to overnight at 55° C. The vial was cooled briefly on ice and then the ethanolic ammonia mixture was transferred to a new microfuge tube. The CPG was washed with 3×0.25 mL portions of 50% acetonitrile (70% CH3CN for cholesterol and such hydrophobic conjugated oligomers). The approximate 1.75 mL of solution is best divided equally into two microfuge tubes, capped tightly and then cooled at −80° C. for 15 min, before drying in a speed vac/lyophilizer for about 90 min.
3. Deprotection-II (Removal of 2′ TBDMS Group)
The white residue obtained was resuspended in 200 μL of triethylamine trihydrofluoride (TEA.3HF, Aldrich) and heated at 65° C. for 1.5h to remove the tertbutyldimethylsilyl (TBDMS) groups at the 2′position. The reaction was then quenched with 400 μL of isopropoxytrimethylsilane (iPrOMe3Si Aldrich) and further incubated on the heating block leaving the caps open for 15 min; (This causes the volatile isopropxytrimethylsilylfluoride adduct to vaporize). The residual quenching reagent was removed by drying in a speed vac. The oligomer was then precipitated in anhydrous methanol (MeOH, 800 μL). The liquid was removed very carefully after spinning in a centrifuge for 5 minutes on the highest speed available. Residual methanol was removed by drying briefly in a speed vac after freezing at −80° C. The crude RNA was obtained as a white fluffy material in the microfuge tube.
4. Quantitation of Crude Oligomer or Raw Analysis
Samples were dissolved in 50% aqueous acetonitrile (0.5 mL) and quantitated as follows: Blanking was first performed with 50% aqueous acetonitrile alone (1 mL).
5 μL of sample and 995 μL of 50% acetonitrile, were mixed well in a microfuge tube, transferred to cuvette and absorbance reading obtained at 260 nm. The crude material is dried down and stored at −20° C.
5. Purification of Oligomers
The crude oligomers were analyzed and purified by HPLC (Mono Q Pharmacia Biotech 5/50). The buffer system is A=100 mM Tris HCl 10% HPLC grade acetonitrile pH=8, B=100 mM Tris-HCl pH 8, 10% HPLC grade acetonitrile 1 M NaCl, flow 1.0 mL/min, wavelength 260 nm. For the unmodified RNA 21mer a gradient of 0-0.6M NaCl is usually adequate. One can purify a small amount of material (˜5 OD) and analyze by CGE or MS. Once the identity of this material is confirmed the crude oligomer can then be purified using a larger amount of material. i.e 40 OD's per run, flow rate of 1 mL/min and a less sensitive wavelength of 280 nm to avoid saturation of the detector. Fractions containing the full length oligonucleotides are then pooled together, evaporated and finally desalted as described below.
6. Desalting of Purified Oligomer
The purified dry oligomer was then desalted using either C-18 Sepak cartridges (Waters) or Sephadex G-25M (Amersham Biosciences). The cartridge was conditioned with 10 mL each of acetonitrile, followed 50% acetonitrile, 100 mM buffer (this can be triethylammonium acetate, sodium acetate or ammonium acetate). Finally the purified oligomer dissolved thoroughly in 10 mL RNAse free water was applied to the cartridge with very slow dropwise elution. The cartridge was washed with water (10 mL) to remove salts. And finally the salt free oligomer was eluted with 50% acetonitrile or 50% methanol directly into a screw cap vial.
7. Capillary Gel Electrophoresis (CGE) and Electrospray LC/Ms
1 μL of approximately 0.04 OD oligomer is first dried down, redissolved in water (2 μL) and then pipetted in special vials for CGE and LC/MS analysis. In general, desalting should be carried out prior to analysis.
TABLE 4
List of RNA oligonucleotides synthesized
siRNA
Sequence
1S
5′-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO: 29)
1AS
5′-UCGAAGUACUCAGCGUAAGdTdT-3′ (SEQ ID NO: 30)
2S
5′-CUUACGCUGAGUACUUCGAUU-3′ (all RNA) (SEQ ID NO: 31)
2AS
5′-UCGAAGUACUCAGCGUAAGUU-3′ (all RNA) (SEQ ID NO: 32)
3S
5′-CUUACGCUGAGUACUUCGAdT*dT-3′ * = PS (SEQ ID NO: 33)
3AS
5′-UCGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 34)
4S
5′-C*UUACGCUGAGUACUUCGAdT*dT-3′ * = PS (SEQ ID NO: 35)
4AS
5′-U*CGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 36)
5S
5′-C*UUACGCUGAGUACUUCGA*dT*dT-3′ * = PS (SEQ ID NO: 37)
5AS
5′-U*CGAAGUACUCAGCGUAAGdT*dT-3′ * = PS (SEQ ID NO: 38)
6S
5′ CUUACGCUGAGUACUUCGAU2′OMeU2′OMe 3′ (SEQ ID NO: 39)
6AS
5′-UCGAAGUACUCAGCGUAAGU2′OMeU2′OMe-3′ (SEQ ID NO: 40)
7S
5′ CUUACGCUGAGUACUUCGAU*2OMeU2′OMe 3′ * = PS (SEQ ID NO: 41)
7AS
5′-UCGAAGUACUCAGCGUAAGU*2′OMeU2′OMe-3′ * = PS (SEQ ID NO: 42)
8S
5′ C*UUACGCUGAGUACUUCGAU*2′OMeU2′OMe 3′ * = PS (SEQ ID NO: 43)
8AS
5′-U*CGAAGUACUCAGCGUAAGU*2′OMeU2′OMe-3′* = PS (SEQ ID NO: 44)
9S
5′-M1CUUACGCUGAGUACUUCGAdTdTM2-3′ (SEQ ID NO: 45)
9AS
5′-M1UCGAAGUACUCAGCGUAAGdTdTM2-3′ (SEQ ID NO: 46)
10S
5′-M1*CUUACGCUGAGUACUUCGAdTdT*M2-3′ (SEQ ID NO: 47)
10AS
5′-M1*UCGAAGUACUCAGCGUAAGdTdT*M2-3′ (SEQ ID NO: 48)
11S
5′-CUUACGCUGAGUACUUCGAdTdTM3-3′ (SEQ ID NO: 49)
11AS
5′-UCGAAGUACUCAGCGUAAGdTdTM3-3′ (SEQ ID NO: 50)
12S
5′-CUUACGCUGAGUACUUCGAdTdT*M3-3′ * = PS (SEQ ID NO: 51)
12AS
5′-UCGAAGUACUCAGCGUAAGdTdT*M3-3′ * = PS (SEQ ID NO: 52)
M1 = 3′-OMe-U, in which the 3′ substituent of the (U) sugar is —OCH3.
M2 = 3′-OMe-U, in which the 3′ substituent of the (U) sugar is —OCH3.
M3 = 3′pyrrolidine cholesterol
* = PS = phosphorothioate linkage
U2′OMe means that the 2′ substituent of the (U) sugar is —OCH3.
dT = deoxythymidine
TABLE 5
Mass data for olignucleotides in Table 4
siRNA
Expected Mass (amu)
LC/MS (amu)
1S
6606.09
6606.67
1AS
6693.06
6692.93
2S
6610.91
6610.68
2AS
6697.01
6696.782
3S
6623.03
6622.76
3AS
6709.13
6708.71
4S
6639.09
4AS
6725.2
6724
5S
6655.16
5AS
6741.26
6740.56
6S
6638.96
6638.66
6AS
6725.06
6724.67
7S
6655.02
6654.57
7AS
6741.13
8S
6671.09
6670.79
8AS
6757.19
6756.84
9S
7247.29
7246.67
9AS
7333.4
7333.11
10S
7263.36
10AS
7349.46
11S
7312.41
7313.06
11AS
7398.51
7397
12S
7328.48
7329
12AS
7414.58
7415.39
Example 12
In Vitro Activity and Cytotoxicity of Chemically Modified siRNAs
Synthetic siRNAs
Firefly luciferase targeting oligoribonucleotides (antisense 5′-UCGAAGUACUCUAGCGUAAGNN-3′) (SEQ ID NO:53) were synthesized and characterized as described above. Twelve unique sense and twelve unique antisense strands were mixed in all possible combinations to yield 144 distinct siRNA duplexes. Sense and antisense strands were arrayed into 96-well PCR plates (VWR, West Chester, Pa.) in annealing buffer (100 mM KOAc, 30 mM HEPES, 2 mM MgOAc, pH 7.4) to give a final concentration of 10 μM duplex. Annealing was performed employing a thermal cycler (ABI PRISM 7000, Applied Biosystems, Foster City, Calif.) capable accommodating the PCR plates. The plates were held at 90° C. for one minute and 37° C. for one hour. Duplex formation was verified by native agarose gel electrophoresis of a random sample of the 144 sense and antisense combinations.
Cell Culture
HeLa SS6 cells were grown at 37° C. in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 □g/mL streptomycin (Invitrogen, Carlsbad, Calif.). Cells were passaged regularly to maintain exponential growth. Twenty-four hours prior to siRNA transfection, cells were seeded on opaque, white 96-well plates (Costar, Corning, N.Y.) at a concentration of 15,000 cells/well in 150 μL antibiotic-free, phenol red-free DMEM (Invitrogen).
Dual Luciferase Gene Silencing Assays
In vitro activity of siRNAs was determined using a high-throughput 96-well plate format luciferase silencing assay. Cells were first transiently transfected with plasmids encoding firefly (target) and renilla (control) luciferase. DNA transfections were performed using Lipofectamine 2000 (Invitrogen) (0.5 μL/μg total DNA) and the plasmids gWiz-Luc (Aldevron, Fargo, N. Dak.) (200 ng/well) and pRL-CMV (Promega, Madison, Wis.) (200 ng/well). After 2 h, the plasmid transfection medium was removed, and the firefly luciferase targeting siRNAs were added to the cells at 100 nM concentration. siRNA transfections were performed using TransIT-TKO (Mirus, Madison, Wis.) (0.3 □L/well). After 24 h, cells were analyzed for both firefly and renilla luciferase expression using a plate luminometer (VICTOR2, PerkinElmer, Boston, Mass.) and the Dual-Glo Luciferase Assay kit (Promega). Firefly/renilla luciferase expression ratios were used to determine percent gene silencing relative to mock-treated (no siRNA) controls.
Cytotoxicity Assays
Cytotoxicity assays were performed in parallel with the gene silencing assays. These assays were carried out in the exact manner as the gene silencing assays (see above) with the exception that 24 h post siRNA tranfection, cells were analyzed for cytotoxicity instead of gene silencing. Relative cell viability was determined by quantification of cellular ATP content using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).
A control and candidate iRNA agents are delineated in FIG. 15.
Relative cell viability results and activity results are represented graphically in FIGS. 16 and 17, respectively. Essentially no activity was observed with duplexes with 12 AS; about 50% activity was observed with 9-11 AS; and full activity was observed with 1-8 AS.
Representative cholesterol-tethered RRMS monomers are shown in FIG. 18. An RRMS monomer having a linked solid support (bottom left) can be incorporated at the 3′ end of an RNA, e.g., an iRNA agent. An RRMS monomer having an amidite (bottom left) can be incorporated at the 5′ end or internal position of an RNA, e.g., an iRNA agent.
LCMS data for a 3′ cholesterol conjugate after PAGE purification is shown in FIG. 19.
Example 13
To evaluate the cell permeation properties of cholesterol conjugated siRNAs 11 sense strand containing 3′ cholesterol conjugate was annealed with 1 antisense strand and applied to the cell culture without any transfection agent. The 1S-1AS duplex was used as an unmodified control. Luciferase expression was silenced by the 11S-1AS duplex with a dose response without the transfection agent, while the unmodified duplex 1S-1AS did not show any gene silencing (see FIG. 20).
Example 14
5′ CHOLESTEROL-CUUACGCUGAGUACUUCGAdTdT-3′ (SEQ ID NO: 54)
Compound 14-a (described e.g., at page 67) was used to synthesize siRNA conjugates where cholesterol was conjugated at the 5′ end of RNA molecules.
The phosphoramidite 14-a was dissolved in acetomitrile/methylene chloride 1:1 solution to give a 0.2M solution. This was used for the terminal coupling during the oligonucleotide synthesis. For the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation Beaucage reagent 0.5M solution in acetomitrile was used. The diamathoxy triotyl group was removed in the synthesizer and the purification and characterization were carried out as described in example 11.
Example 15
Additional Ligand-Conjugated Monomer Syntheses
Scheme and compound numbers refer to those recited in Example 15.
Synthesis of 4-Hydroxy-L-Prolinol Linker
1-(6-Benzyloxycarbonylamino-hexanoyl)-4-hydroxy-pyrrolidine-2-carboxylic Acid Methyl Ester (2a)
Referring to scheme 1, 6-benzyloxyamino hexanoic acid (13.25 g, 50 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50 mmol) and triethylamine (10.2 g, 13.7 mL, 100 mmol). After stirring for 20 mins at 0° C., 4-hydroxy-L-proline methyl ester hydrochloride (9.6 g, 50 mmol) was added and the stirring was continued at room temperature under argon for over night. The reaction mixture was evaporated to dryness. To the residue ethyl acetate (100 mL) was added and the filtered to remove diisopropyl urea. The precipitate was washed with ethyl acetate (50 mL). The combined organic layer was washed with 2N HCl, saturated sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. Compound 2a (Rf=0.6 in 10% MeOH/CHCl3, 22 g) was obtained, which was directly used for the next step without further purification.
[6-(4-Hydroxy-2-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic Acid Benzyl Ester (3a)
To the solution of lithium borohydride (1.34 g) in anhydrous tetrahydrofuran (50 mL) was added a solution of methyl ester 2a in THF (50 mL) over a period of 30 mins at 0° C. After the addition the reaction mixture was brought to room temperature and stirred further under argon. The completion of the reaction was ascertained by TLC after 4 h. (Rf=0.4 in 10% MeOH/CHCl3). The reaction mixture was evaporated to dryness and cooled to 0° C. To the residue 3N HCl (100 mL) was added slowly. After stirring for 30 mins the product was extracted with dichloromethane (3×100 mL). The combined organic layer was washed with brine and dried over sodium sulfate. Organic layer was filtered and evaporated to dryness. Compound 3a was purified by column chromatography first by eluting with ethyl acetate to remove impurities followed by dichloromethane/methanol (5%) gave 14.3 g (70%)
1H NMR (400 MHz, DMSO-d6): Observed rotamers due to amide bond at the ring. δ 7.35 (m, 5H), 5.0 (s, 2H), 4.92 (d, OH, D2O exchangeable, 4.78 (t, OH, D2O exchangeable) 4.28 (m, 1H), 3.95 (m, 1H), 3.2-3.48 (m, 5H), 2.92-3.0 (m, 2H), 2.1-2.3 (m, 2H), 1.7-2.0 (2H), 1.34-1.52 (m, 4H), 1.2-1.3 (m, 2H).
13C NMR (100 MHz, DMSO-d6): δ171.3, 171.1 (minor due to rotamer which disappears while performing at 80° C.), 156.1, 137.3, 128.3, 127.7, 68.2, 65.1, 61.9, 57.5, 55.1, 36.1, 34.2, 29.3, 26.1, 25.9, 24.6, 24.1, 20.77, 14.09.
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic Acid Benzyl Ester (4a)
Referring to scheme 1, compound 3a (14 g, 38.4 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (60 mL). To this solution dimethylamino pyridine (0.488 g, 4 mmol) and DMT-Cl (13.6 g, 40.3 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The excess DMT-Cl was quenched by the addition of methanol (25 mL). The solution was dried under reduced pressure. To the residue was suspended in ethyl acetate (300 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. 24.2 g of the crude product was obtained after removal of the solvent. Upon purification over silica gel using 2% MeOH/DCM compound 4a (21.3 g, 83%) was obtained as white foamy solid.
1H NMR (400 MHz, DMSO-d6): δ 7.18-7.38 (m, 14H), 6.2-6.5 (m, 4H), 5.0 (s, 2H), 4.9 (d, —OH, D2O exchangeable), 4.4 (m, 1H), 4.15 (m, 1H), 3.7 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 2.9-3.0 (m, 4H), 2.18 (m, 2H), 1.8-2.1 (m, 2H), 1.1-1.5 (m, 6H).
13C NMR (100 MHz, CDCl3): δ 174.7, 172.7, 171.9, 171.3, 171.2, 158.8, 158.7, 158.6, 158.5, 158.4, 158.3, 156.7, 156.7, 156.6, 147.5, 145.8, 145.2, 144.9, 144.7, 144.4, 139.6, 137.1, 137.04, 137.01, 136.9, 136.82, 136.78, 136.55, 136.47, 136.45, 136.3, 136.28, 135.93, 135.85, 135.81, 130.2, 130.1, 130.0, 129.9, 129.3, 128.69, 128.66, 128.22, 128.16, 128.0, 127.99, 127.94, 127.91, 127.77, 113.52, 113.43, 113.35, 113.3, 113.24, 113.19, 113.03, 86.8, 86.1, 85.9, 73.0, 71.6, 71.5, 70.5, 69.3, 67.3, 67.1, 68.76, 68.71, 64.38, 63.7, 60.58, 60.0, 56.4, 55.8, 55.7, 55.45, 55.41, 55.35, 55.33, 40.97, 40.87, 40.77, 37.13, 36.83, 35.13, 35.00, 34.81, 34.6, 33.3, 29.8, 26.73, 25.5, 26.4, 26.2, 24.9, 24.6, 24.5, 24.3, 24.2, 21.1, 14.3.
6-Amino-1-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexan-1-one (5)
Compound 4a (14.5 g, 21.7 mmol) was dissolved in ethyl acetate (100 mL) and purged with argon. To the solution was added 10% palladium on carbon (2 g). The flask was purged with hydrogen 2 times and stirred further at room temperature under hydrogen atmosphere for overnight. The disappearance of the starting material was confirmed by the TLC. The reaction mixture was filtered through a pad of Celite and washed with ethyl acetate. The combined organic layer was concentrated under reduced pressure to afford compound 5 (10.56 g, 91%) as white solid. This was used as such for the next step.
1H NMR (400 MHz, DMSO-d6): δ 7.16-7.32 (m, 9H), 6.86 (m, 4H), 5.0 (bs, 1H), 4.4 (m, 1H), 3.9-4.25 (m, 2H), 3.72 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 2.98-3.0 (m, 2H), 2.45 (m, 2H), 2.2 (m, 2H), 1.8-2.04 (m, 3H), 1.1-1.45 (m, 4H).
13C NMR (100 MHz, DMSO-d6): δ 17.9, 157.9, 145.1, 144.76, 135.8 135.7, 129.5, 127.8, 127.7, 127.5, 126.5, 113.2, 113.1, 85.7, 85.0, 68.5, 67.4, 63.3, 54.9, 41.6, 36.2, 34.2, 33.3, 32.5, 26.2, 24.7, 24.4.
Compound 4b:
The desired compound 4b is obtained from NCbz-12-aminododecanoic acid (1b) and trans-4-hydroxyproline methyl ester hydrochloride in three steps as described for the synthesis of compound 4a from compound 1a.
Synthesis of 4-hydroxy-L-prolinol Cholesterol Phosphoramidite
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester (6)
Referring to scheme 2, compound 5 (13.3 g, 25 mmol) was dissolved in anhydrous dichloromethane (100 mL) and cooled to 0° C. To the solution were added triethylamine (7.5 g, 10 mL, 75 mmol) and cholesteryl chloroformate (11.24 g, 25 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 2h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was evaporated under the vacuum to afford the crude product. Compound 6 (22.1 g, 93%) was obtained as a white foamy solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 7.12-7.3 (m, 8H), 6.95 (m, 1H), 6.84 (m, 4H), 5.3 (bs, 1H), 4.92 and 4.84 (d, OH, exchangeable with D2O), 4.21-4.38 (m, 2H), 4.35 (m, 1H), 3.7 (s, 6H), 3.54 (m, 1H), 3.28 (m, 2H), 3.12 (m, 1H), 2.84-2.98 (m, 3H), 2.12-2.28 (m, 3H), 1.7-2.0 (m, 7H), 0.8-1.52 (m, 40H), 0.6 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7, 135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1, 72.7, 68.5, 63.3, 56.1, 55.5, 54.9, 49.4, 41.8, 36.5, 35.2, 31.3, 27.7, 27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3, 20.5, 18.9, 18.5, 11.6.
4-hydroxy-L-prolinol-cholesterol-phosphoramidite (7)
Compound 6 (4.0 g, 4.23 mmol) was coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g, 2.1 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.5 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 7 as white foam (4.35 g, 89%).
1H NMR (400 MHz, CDCl3): δ 7.14-7.38 (m, 9H), 6.8 (m, 4H), 5.36 (bs, 1H), 4.34-4.7 (m, 4H), 3.4-3.82 (m, 13H), 3.15 (m, 3H), 2.58 (m, 2H), 1.8-2.38 (m, 12H), 0.84-1.68 (m, 49H), 0.76 (s, 3H).
31P NMR (161.82 MHz, CDCl3): δ 145.9, 145.7, 145.4, 145.0 (1:2 ratio, 4 peaks due to rotamers).
13C NMR (100 MHz, CDCl3): δ 171.8, 158.7, 158.5, 156.3, 145.3, 144.7, 140.1, 136.4, 136.36, 136.32, 135.8, 130.1, 129.2, 128.4, 128.27, 128.21, 128.13, 127.9, 127.1, 126.9, 125.5, 122.6, 111.8, 117.7, 113.4, 113.2, 86.16, 86.1, 74.3, 72.3, 58.5, 58.3, 58.1, 56.8, 56.3, 55.9, 55.8, 55.4, 55.3, 52.2, 43.4, 43.3, 42.5, 40.8, 39.9, 39.7, 38.7, 37.2, 36.7, 36.3, 36.0, 35.0, 32.1, 32.0, 30.0, 28.45, 28.4, 28.2, 26.8, 24.8, 24.7, 24.69, 24.6, 24.5, 24.0, 23.0, 22.7, 21.6, 21.2, 20.6, 20.59, 20.52, 19.5, 18.9, 12.0
Synthesis of Solid Support with Immobilized Cholesterol
Succinic Acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino)-hexanoyl]-pyrrolidin-3-yl} Ester (8)
Referring to scheme 3, Compound 6 (22 g, 23.2 mmol) was mixed with succinic anhydride (3.48 g, 34.8 mmol) and DMAP (0.283 g, 2.32 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (50 mL), triethylamine (7 g, 9.6 mL, 70 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 8 as white solid (21.7 g, 89% yield; Rf=0.5 in 10% MeOH/CHCl3).
1H NMR (400 MHz, CDCl3): δ 7.32-7.36 (m, 2H), 7.2-7.28 (m, 7H), 6.76-6.8 (m, 4H), 5.4 (bs, 1H), 4.46 (m, 2H), 3.78 (s, 6H), 3.42 (m, 1H), 3-3.18 (m, 3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 0.8-1.6 (m, 42H), 0.66 (s, 3H)
13C NMR (100 MHz, CDCl3): δ 176.59, 172.22, 158.78, 158.62, 145.16, 139.8, 136.39, 136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91, 113.28, 56.88, 56.32, 55.45, 55.4, 50.19, 45.47, 42.51, 39.93, 39.72, 38.67, 37.14, 36.74, 36.38, 36.0, 32.1, 32.06, 28.44, 28.22, 24.5, 24.0, 23.04, 22.77, 21.24, 19.55, 18.92, 12.07, 8.72
Solid Support Immobilized with Cholesterol (9)
Succinate 8 (10.45 g, 10 mmol) was dissolved in dichloroethane (50 mL). To that solution DMAP (1.22 g, 10 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (3.1 g, 10 mmol) in acetonitrile/dichloroethane (3:1, 50 mL) was added successively. To the resulting solution triphenylphosphine (2.63 g, 10 mmol) in acetonitrile (25 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (70 g, 155 μm/g) was added. The suspension was agitated for 16 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (62 μM/g).
Synthesis of 4-hydroxy-L-prolinol-rac-dioctadecy Glyceryl Amidite
1,2-Di-O-octadecyl-rac-glycerol Succinimidyl Carbamate (11)
Referring to scheme 4, 1,2-Di-O-octadecyl-rac-glycerol (10 g, 16.74 mmol) was dissolved in anhydrous dichloromethane (150 mL). To the solution were added disuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine (10 mL) and acetonitrile (50 mL). The reaction mixture was stirred at room temperature under argon for 6h and then evaporated dryness. The residue was dissolved in dichloromethane (300 mL). It was washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Compound 11 (12.8 g) was obtained as colorless powder after drying in high vacuum, which was directly used for the next step without further purification.
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 2,3-bis-octadecyloxy-propyl Ester (2)
Amine 5 (10.5 g, 19.7 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL) and compound 11 (12.5 g, 17.3 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 3h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 12 (17.8 g, 89%) was obtained as a white solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 7.2-7.38 (m, 9H), 6.76 (m, 4H), 5.4 (s, 3H), 4.0 (m, 2H), 3.25 (s, 6H), 2.96 (m, 2H), 2.0 (m, 3H), 3-3.18 (m, 3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 1.2-1.6 (m, 76H), 0.8 (m, 3H)
13C NMR (100 MHz, DMSO-d6): δ 171.89, 171.38, 158.74, 158.56, 156.70, 156.6, 145.28, 144.77, 136.49, 136.33, 135.89, 135.8, 130.19, 130.15, 128.25, 128.20, 128.09, 127.94, 127.13, 126.90, 113.37, 113.21, 86.68, 86.05, 71.98, 70.8, 70.69, 70.61, 69.40, 65.6, 64.38, 63.8, 60.61, 40.93, 38.48, 36.97, 35.0, 33.3, 32.13, 31.21, 29.9, 29.86, 29.72, 29.56, 26.59, 26.30, 26.24, 24.66, 22.89, 21.26, 14.39, 14.33
4-hydroxy-L-prolinol-rac-dioctadecy Glyceryl Amidite (13)
Compound 12 (10.0 g, 8.65 mmol) was coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.488 g, 4.32 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (50 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (3.91 g, 4.28 mL, 13 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.6 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 13 as white solid (15.37 g, 93%).
1H NMR (400 MHz, CDCl3): δ 7.16-7.38 (m, 9H), 6.78 (m, 4H), 4.62-4.78 (m, 2H), 4.27 (m, 1H), 4.04-4.2 (m, 3H), 3.7-3.8 (m, 10H), 3.4-3.6 (m, 11H), 3.16 (m, 4H), 2.58-2.7 (m, 4H), 2.22 (m, 3H), 2.12 (m, 1H), 1.15-1.4 (m, 75H), 0.95 (m, 6H),
31P NMR (161.82 MHz, CDCl3): δ 145.96, 145.76, 145.45, 145.07
13C NMR (100 MHz, CDCl3): δ 171.79, 171.61, 158.75, 158.58, 156.59, 145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22, 130.19, 128.28, 128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24, 86.11, 71.98, 70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69, 64.37, 63.92, 58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39, 46.31, 44.70, 44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23, 29.92, 29.88, 29.72, 29.58, 26.32, 26.26, 24.85, 24.78, 24.68, 22.9, 20.58, 14.34
Synthesis of Solid Support with Immobilized Rac-Dioctadecy Glycerol (15)
Succinic Acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(2,3-bis-octadecyloxy-propoxycarbonylamino)-hexanoyl]-pyrrolidin-3-yl} Ester (14)
Referring to scheme 5, Compound 12 (5.6 g, 4.8 mmol) was mixed with succinic anhydride (0.727 g, 7.26 mmol) and DMAP (0.062 g, 0.5 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (20 mL), triethylamine (1.52 g, 2 mL, 15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 50 mL) and water (2×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 14 as white solid (2.85 g, 47% yield; Rf=0.65 in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 12.2 (bs, 1H), 7.18-7.4 (m, 9H), 6.82 (m, 4H), 4.62-4.78 (m, 2H), 4.27 (m, 1H), 4.04-4.2 (m, 3H), 3.7-3.8 (m, 10H), 3.4-3.6 (m, 11H), 3.16 (m, 4H), 2.58-2.7 (m, 4H), 2.22 (m, 3H), 2.12 (m, 1H), 1.15-1.4 (m, 75H), 0.95 (m, 6H),
13C NMR (100 MHz, DMSO-d6): δ 178.26, 174.23, 171.79, 171.61, 158.75, 158.58, 156.59, 145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22, 130.19, 128.28, 128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24, 86.11, 71.98, 70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69, 64.37, 63.92, 58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39, 46.31, 44.70, 44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23, 29.92, 29.88, 29.72, 29.58, 28.41, 26.32, 26.26, 24.85, 24.78, 24.68, 22.9, 20.58, 14.34.
Solid Support with Immobilized Rac-Dioctadecy Glycerol (15)
Succinate 14 (2 g, 1.6 mmol) was dissolved in dichloroethane (8 mL). To that solution DMAP (0.194 g, 1.6 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.496 g, 1.6 mmol) in acetonitrile/dichloroethane (3:1, 8 mL) was added successively. To the resulting solution triphenylphosphine (0.419 g, 1.6 mmol) in acetonitrile (4 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (5.16 g, 800 μmoles, 155 μm/g) was added.
The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (76 μM/g).
Synthesis of 4-hydroxy-L-prolinol-vitamin E Amidite
2-(2-Hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethanol (17)
Referring to scheme 6, vitamin E (16.0 g, 37 mmol) was dissolved in acetone (100 mL). Potassium carbonate (25.5 g, 185 mmol), ethylene carbonate (6.5 g, 75 mmol) were added to the solution. The suspension was stirred at reflux temperature for over night. Even though the reaction did not go to completion, the reaction mixture was concentrated in the vacuum, and the residue was taken in ethyl acetate and washed with water. The organic layer was dried over sodium sulfate, filtered and evaporated. The crude product was purified by column chromatography using hexane/ethyl acetate to afford compound 17 in 65% yield (11.5 g, Rf=0.8 in 25% EtOAc/Hexane).
1H NMR (400 MHz, CDCl3): δ 4.13 (m, 2H), 3.98 (m, 2H), 2.6 (t, 2H), 2.15 (s, 3H), 2.1 (s, 6H), 1.7-1.8 (m, 2H), 1.1-15 (m, 14H), 0.8-0.88 (m, 12H)
13C NMR (100 MHz, CDCl3): δ 145.75, 144.74, 122.83, 121.2, 118.66, 117.58, 77.43, 74.74, 60.63, 40.08, 40.01, 39.59, 37.80, 37.79, 37.71, 37.67, 37.6, 37.55, 37.50, 33.01, 33.0, 32.91, 31.8, 31.57, 31.69, 28.20, 25.04, 25.02, 24.66, 24.01, 22.95, 22.85, 21.28, 20.98, 19.97, 19.9, 19.86, 19.81, 14.42, 14.35, 12.43, 12.0, 11.5
Carbonic acid 2,5-dioxo-pyrrolidin-1-yl ester 2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethyl Ester (18)
Compound 17 (10.5 g, 22 mmol) was dissolved in anhydrous dichloromethane (150 mL). To the solution were added disuccinimidyl carbonate (8.45 g, 33 mmol), triethylamine (20 mL) and acetonitrile (50 mL). The reaction mixture was stirred at room temperature under argon for over night and then evaporated dryness. The residue was dissolved in dichloromethane (300 mL). It was washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Compound 18 (10.4 g, 77%) was obtained as colorless powder after drying in high vacuum, which was directly used for the next step without further purification.
1H NMR (400 MHz, CDCl3): δ 7.2-7.4 (m, 4H), 3.1 (m, 1H), 2.7 (s, 4H), 2.58 (t, 2H), 2.08-2.14 (m, 9H), 1.7-1.82 (m, 2H), 1-1.6 (m, 23H), 0.8-0.88 (m, 12H)
13C NMR (100 MHz, CDCl3): δ 168.76, 168.66, 150.6, 150.37, 141.36, 138.09, 129.24, 128.44, 126.73, 125.51, 125.1, 123.74, 117.97, 77.72, 75.56, 39.58, 37.71, 37.65, 37.59, 37.48, 32.98, 32.88, 31.16, 31.11, 28.19, 25.83, 25.75, 25.7, 25.02, 25.01, 24.64, 24.07, 24.07, 22.93, 22.84, 21.67, 21.22, 20.73, 20.38, 19.96, 19.89, 19.86, 19.82, 12.77, 11.98, 11.94
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic Acid 2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethyl Ester (19)
Amine 5 (8.7 g, 16.3 mmol) was dissolved in anhydrous dichloromethane (40 mL) and cooled to 0° C. To the solution were added triethylamine (5.06 g, 6.73 mL, 50 mmol) and compound 18 (10 g, 16.2 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 6h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 19 (14.5 g, 88%) was obtained as a white foamy solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 7.72 (m, 1H, —NH), 7.3 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (s, —OH), 4.38 (m, 2H), 4.12 (m, 2H), 3.72 (s, 6H), 3.56 (m, 1H), 3.22-3.32 (m, 2H), 3.16 (m, 1H), 3.0 (m, 3H), 2.2 (m, 2H), 1.98 (m, 4H), 1-9 (m, 7H), 1.8 (m, 1H), 1.72 (m, 2H), 1-1.5 (m, 32H), 0.82 (m, 12H)
13C NMR (100 MHz, DMSO-d6): δ 171.20, 158.29, 158.16, 154.83, 148.31, 145.26, 140.65, 136.04, 135.90, 135.58, 129.78, 127.97, 127.75, 127.43, 126.79, 125.91, 121.58, 117.10, 113.28, 86.0, 85.32, 74.71, 68.71, 63.46, 55.13, 36.91, 36.8, 36.35, 32.25, 27.75, 26.06, 24.36, 23.91, 23.68, 22.68, 22.59, 19.75, 19.68, 12.74, 11.89, 11.64
4-hydroxy-L-prolinol-vitamin E Amidite (20)
Compound 19 (9.2 g, 9 mmol) was coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.51 g, 4.5 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (50 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (4 g, 4.45 mL, 13.5 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.65 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 20 as white foamy solid (10.5 g, 95%).
1H NMR (400 MHz, CDCl3): δ 7.38 (m, 2H), 7.18-7.28 (m, 7H), 6.82 (m, 4H), 5.18 (m, 1H), 4.65 (m, 2H), 4.38 (m, 2H), 4.1 (m, 1H), 3.7-3.8 (m, 9H), 3.58 (m, 3H), 3.4 (m, 1H), 3.28 (m, 2H), 3.18 (m, 2H), 2.58 (m, 4H), 2.26 (m, 3H), 2-2.1 (m, 10H), 1.5-1.8 (m, 10H), 1.05-1.3 (m, 32H), 0.84-0.88 (m, 14H).
31P NMR (161.82 MHz, CDCl3): δ 145.92, 145.78, 145.45, 145.04
13C NMR (100 MHz, CDCl3): δ 172.1, 171.82, 171.63, 158.74, 158.67, 155.17, 149.34, 145.31, 144.77, 140.58, 136.44, 136.34, 136.3, 135.85, 135.8, 130.18, 128.25, 128.19, 128.11, 127.95, 127.82, 127.13, 126.90, 126.0, 123.0, 117.87, 117.78, 117.42, 113.4, 113.29, 113.23, 86.77, 86.15, 86.10, 77.42, 75.15, 72.39, 72.21, 72.01, 63.92, 58.65, 58.53, 58.47, 58.35, 58.13, 56.49, 55.96, 55.85, 55.44, 55.37, 54.65, 43.44, 43.31, 41.22, 40.33, 39.55, 37.74, 37.64, 37.58, 37.47, 35.06, 33.44, 32.97, 32.9, 31.3, 30.1, 28.17, 26.7, 26.74, 26.68, 25.12, 24.99, 24.84, 24.77, 24.64, 24.57, 24.11, 22.92, 22.83, 21.23, 21.25, 20.76, 20.63, 20.56, 20.50, 19.95, 19.88, 19.84, 19.82, 19.78, 13.05, 12.20, 11.97.
Synthesis of Solid Support with Immobilized Vitamin E (22)
Succinic Acid mono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[2-(2-hexadecyl-2,5,7,8-tetramethyl-chroman-6-yloxy)-ethoxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) Ester (21)
Referring to scheme 7, Compound 19 (5.1 g, 5 mmol) was mixed with succinic anhydride (0.75 g, 7.5 mmol) and DMAP (0.062 g, 0.5 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (25 mL), triethylamine (1.52 g, 2 mL, 15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 50 mL) and water (2×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 21 as white foamy solid (2.85 g, 51% yield; Rf=0.65 in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 12.3 (bs, 1H), 7.6 (m, 1H), 7.2-7.4 (m, 9H), 6.86 (m, 4H), 5.32 (m, 2H), 4.18 (m, 2H), 3.62-3.8 (s, 6H), 3.54 (m, 1H), 3.42 (m, 1H), 3.34 (s, 6H), 3.21 (m, 1H), 3.0 (m, 2H), 2.46 (m, 4H), 2.2 (m, 4H), 1.9 (m, 4H), 1.72 (m, 3H), 1-1.5 (m, 30H), 0.82 (m, 12H).
13C NMR (100 MHz, DMSO-d6): δ 173.61, 172.26, 171.17, 165.43, 159.77, 158.34, 158.22, 157.02, 154.88, 153.72, 148.68, 148.36, 145.17, 144.85, 143.74, 141.94, 140.63, 135.93, 129.83, 128.06, 127.77, 127.46, 125.97, 121.63, 117.25, 113.35, 85.50, 74.86, 73.08, 55.19, 36.88, 32.17, 28.97, 28.80, 27.58, 24.34, 23.85, 22.75, 22.66, 19.84, 19.78, 12.8, 11.94, 11.72.
Solid Support with Immobilized Vitamin E (22)
Succinate 21 (2.8 g, 2.5 mmol) was dissolved in dichloroethane (12 mL). To that solution DMAP (0.306 g, 2.5 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.775 g, 2.5 mmol) in acetonitrile/dichloroethane (3:1, 12 mL) was added successively. To the resulting solution triphenylphosphine (0.656 g, 2.5 mmol) in acetonitrile (7 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (8.0 g, 1240 μmoles, 155 μm/g) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG 22 was measured by taking UV measurement. (76 μM/g).
Synthesis of 4-hydroxy-L-prolinol-thicholesterol Amidite (26)
N-(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-3-(pyridin-2-yldisulfanyl)-propionamide (24)
Referring to scheme 8, amine 5 (7.7 g, 14.5 mmol) was dissolved in anhydrous dichloromethane (40 mL) and cooled to 0° C. To the solution were added triethylamine (3.0 g, 4.2 mL, 30 mmol) and 3-(Pyridin-2-yldisulfanyl)-propionic succinate ester 23 (SPDP) (4.5 g, 14.4 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 16h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3, Rf=0.6). The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 24 (10.58 g, 78%) was obtained as a white foamy solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 8.45 (d, 1H), 7.9 (m, 1H), 7.8 (m, 1H), 7.76 (m, 1H), 7.3 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (d, —OH, 1H), 4.38 (m, 1H), 4.1 (m, 1H) (s, 6H), 3.56 (m, 1H), 3.46 (m, 1H), 3.21-3.34 (m, 3H), 3.14 (m, 1H), 3 (m, 2H), 2.48 (m, 2H), 2.2 (m, 2H), 1.8-2.02 (m, 2H), 1.1-1.5 (4H).
13C NMR (100 MHz, DMSO-d6): δ 171.32, 169.97, 159.36, 158.31, 158.18, 149.80, 145.27, 138.08, 136.1, 135.9, 129.8, 128.0, 127.7, 121.4, 119.3, 113.3, 85.338, 68.7, 55.3, 34.75, 34.28, 29.1, 26.3, 24.36.
4-Hydroxy-L-prolinol-thiocholesterol-DMT-alcohol 25
Compound 24 (7.5 g, 10.28 mmol) was dissolved in anhydrous dichloromethane (75 mL) under argon and cooled to 0° C. To this solution were added diisopropylethyl amine (2.71 g, 3.66 mL, 21 mmol) followed by thiocholesterol (4.145 g, 10.28 mmol). The reaction mixture was brought to ambient temperature and stirred further for 16 h. The completion of the reaction was ascertained by TLC (100% ethyl acetate, Rf=0.6). The reaction mixture was concentrated under reduced pressure and the residue was subjected to column chromatography on silica gel. Even though there was good separation in hexane/ethyl acetate system, compound precipitates in that mixture. After eluting with 4 L of ethyl acetate, the column was eluted with 5% MeOH/dichloromethane (2 L) to obtain compound 25 as white foamy solid (8 g, 76%).
1H NMR (400 MHz, DMSO-d6): δ 7.88 (m, 1H), 7.3 (m, 4H), 7.17 (m, 5H), 6.84 (m, 4H), 5.3 (bs, 1H), 4.89 (d, —OH), 4.38 (m, 1H), 4.1 (m, 1H), 3.72 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 3 (m, 3H), 2.84 (m, 2H), 2.64 (m, 1H), 2.42 (m, 2H), 2.2 (m, 3H), 1.8-2.0 (m, 7H), 0.8-1.54 (m, 35H), 0.62 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7, 135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1, 72.7, 68.5, 63.3, 60.72, 56.1, 55.5, 55.28, 54.9, 49.4, 41.8, 36.5, 35.2, 31.3, 30.35, 27.7, 27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3, 21.11, 20.5, 19.43, 18.9, 18.5, 14.4, 11.6.
4-hydroxy-L-prolinol-thiocholesterol Phosphoramidite (26)
Compound 25 (5.7 g, 5.58 mmol) was coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.315 g, 2.79 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (20 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (2.48 g, 2.72 mL, 8.25 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC (Rf=0.9 in ethyl acetate). The reaction mixture was diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 26 as white foamy solid (6.1 g, 89%).
1H NMR (400 MHz, C6D6): δ 7.62 (m, 2H), 7.45 (m, 5H), 7.24 (m, 2H), 7.1 (m, 1H), 6.82 (m, 4H), 5.64 (m, 1H), 5.38 (m, 1H), 4.7 (m, 1H), 4.54 (m, 2H), 3.78 (m, 2H), 3.5 (m, 3H), 3.36 (m, 9H), 3.22 (m, 4H), 3.06 (m, 3H), 2.72 (m, 1H), 2.32-2.54 (m, 5H), 1.8-2.2 (m, 10H), 1.08-1.74 (m, 28H), 1.3 (m, 6H), 0.94 (m, 12H), 0.67 (s, 3H).
31P NMR (161.82 MHz, C6D6): δ 146.05, 145.91, 145.66, 145.16
13C NMR (100 MHz, C6D6): δ 171.43, 171.25, 169.87, 159.25, 159.11, 146.08, 141.59, 136.66, 136.6, 130.62, 130.54, 128.63, 127.53, 127.02, 121.53, 117.73, 117.57, 113.66, 113.57, 86.59, 86.54, 64.36, 58.56, 58.37, 58.30, 56.96, 56.51, 56.07, 54.86, 54.77, 50.57, 50.27, 43.48, 43.35, 42.55, 40.13, 39.9, 39.75, 39.56, 38.70, 36.94, 36.64, 36.29, 36.19, 35.90, 34.58, 32.24, 32.08, 29.48, 29.03, 28.98, 28.6, 28.38, 26.54, 24.68, 24.61, 24.54, 23.6, 23.0, 22.74, 21.26, 20.03, 19.9, 19.38, 19.01, 12.06.
Synthesis of Polymer Support Immobilized with Thiocholesterol 28
4-Hydroxy-L-prolinol-thiocholesterol-succinate 27
Referring to scheme 9, Compound 25 (2.2 g, 2.15 mmol) was mixed with succinic anhydride (0.323 g, 3.23 mmol) and DMAP (0.026 g, 0.215 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (10 mL), triethylamine (0.708 g, 0.976 mL, 7 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) and water (2×25 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 27 as white foamy solid (2.2 g, 92% yield; Rf=0.6s in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 12.22 (bs, 1H), 7.84 (m, 1H), 7.25 (m, 4H), 7.2 (m, 5H), 6.86 (m, 4H), 5.36 (m, 2H), 4.18 (bs, 1H), 3.72 (s, 6H), 3.4-3.6 (m, 2H), 3.2 (m, 1H), 3.0 (m, 4H), 2.84 (m, 2H), 2.64 (m, 2H), 2.4-2.52 (m, 12H), 2.2 (m, 6H), 1.9 (m, 8H), 0.8-1.52 (m, 28H), 0.65 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 173.35, 171.94, 170.63, 169.64, 157.99, 144.96, 141.02, 135.72, 129.61, 127.81, 127.55, 113.12, 56.15, 54.99, 52.28, 49.58, 49.06, 41.82, 36.17, 34.97, 33.41, 33.09, 31.32, 27.39, 23.16, 22.68, 22.39, 20.56, 18.95, 18.54, 11.66, 6.02, 5.0
Solid Support with Immobilized Thiocholesterol (28)
Succinate 27 (2.1 g, 1.9 mmol) was dissolved in dichloroethane (8 mL). To that solution DMAP (0.228 g, 1.9 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.58 g, 1.9 mmol) in acetonitrile/dichloroethane (3:1, 8 mL) was added successively. To the resulting solution triphenylphosphine (0.49 g, 1.9 mmol) in acetonitrile (4 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (12 g, 1860 μmoles, 155 μm/g) was added.
The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG 28 was measured by taking UV measurement. (57 μM/g).
Synthesis of 4-hydroxy-L-prolinol cholesterol-phosphoramidite (N-alkyl Linkage) (33)
[6-(4-Hydroxy-2-hydroxymethyl-pyrrolidin-1-yl)-hexyl]-carbamic Acid Benzyl Ester (29)
Referring to scheme 10, compound 3a (7 g, 19.2 mmol) was dissolved in anhydrous THF and cooled to 0° C. under argon atmosphere. Borane-THF (50 mL, 1M soln. in THF, 2.5 equiv.) was added slowly over a period of 15 mins. The reaction mixture was brought to room temperature and stirred at reflux temperature for over night. After 16 h, the reaction mixture was cooled and concentrated under vacuum to dryness. To the residue, saturated solution of ammonium chloride (200 mL) was added and the product extracted with ethyl acetate (3×100 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude was purified by column chromatography over silica gel to afford compound 29 as a viscous liquid (6.2 g, 92%).
1H NMR (400 MHz, DMSO-d6): δ 7.33 (m, 5H), 5.1 (s, 2H), 4.94 (d, OH, D2O exchangeable, 4.76 (t, OH, D2O exchangeable) 3.68 (m, 1H), 3.95 (m, 2H), 2.92-3.0 (m, 4H), 2.1-2.3 (m, 3H), 1.7-2.0 (2H), 1.34-1.52 (m, 6H), 1.2-1.3 (m, 4H).
13C NMR (100 MHz, DMSO-d6): 156.1, 137.3, 128.3, 127.7, 68.2, 65.1, 61.9, 57.5, 56.2, 55.1, 36.1, 34.2, 29.3, 26.1, 25.9, 24.6, 24.1.
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexyl)-carbamic Acid Benzyl Ester (30)
Compound 29 (6 g, 17 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (60 mL). To this solution dimethylamino pyridine (0.207 g, 1.7 mmol) and DMT-Cl (6 g, 17.9 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The excess DMT-Cl was quenched by the addition of methanol (25 mL). The solution was dried under reduced pressure. To the residue was suspended in ethyl acetate (300 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. 24.2 g of the crude product was obtained after removal of the solvent. Upon purification over silica gel using 2% MeOH/DCM compound 30 (8.7 g, 79%) was obtained as white foamy solid.
1H NMR (400 MHz, DMSO-d6): δ 7.18-7.38 (m, 14H), 6.2-6.5 (m, 4H), 5.0 (s, 2H), 4.9 (d, —OH, D2O exchangeable), 4.4 (m, 1H), 4.15 (m, 1H), 3.7 (s, 6H), 3.56 (m, 1H), 3.32 (m, 1H), 3.14 (m, 1H), 2.9-3.0 (m, 6H), 2.18 (m, 2H), 1.8-2.1 (m, 2H), 1.1-1.5 (m, 6H).
1-(6-Amino-hexyl)-5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol (31)
Compound 30 (6.52 g, 10 mmol) was dissolved in ethyl acetate (100 mL) and purged with argon. To the solution was added 10% palladium on carbon (2 g). The flask was purged with hydrogen 2 times and stirred further at room temperature under hydrogen atmosphere for overnight. The disappearance of the starting material was confirmed by the TLC. The reaction mixture was filtered through a pad of Celite and washed with ethyl acetate. The combined organic layer was concentrated under reduced pressure to afford compound 31 (4.8 g, 93%) as white solid. This was used as such for the next step.
(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexyl)-carbamic Acid 10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester (32)
Compound 31 (4.5 g, 8.67 mmol) was dissolved in anhydrous dichloromethane (100 mL) and cooled to 0° C. To the solution were added triethylamine (2.52 g, 3.36 mL, 25 mmol) and cholesteryl chloroformate (3.89 g, 8.67 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 2h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was evaporated under the vacuum to afford the crude product. Compound 32 (3.05 g, 37%) was obtained as a white foamy solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 7.1-7.4 (m, 9H), 6.8 (m, 4H), 5.25 (b, 1H), 4.65 (s, 1H), 5.35 (bs, 1H), 4.05 (m, 1H), 3.65 (s, 6H), 3.32 9s, 1H), 3.14 (m, 2H), 2.6-2.9 (m, 8H), 2-2.2 (m, 4H), 0.6-1.8 (m, 48H).
13C NMR (100 MHz, DMSO-d6): 157.922, 148.38, 140.26, 138.89, 129.78, 129.02, 127.74, 127.55, 112.87, 85.41, 67.72, 59.91, 55.1, 54.97, 54.83, 22.53, 22.34, 20.87, 19.22, 14.18.
4-hydroxy-L-prolinol-cholesterol-phosphoramidite (N-alkyl Linkage) (33)
Compound 32 (2.0 g, 2.14 mmol) was coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.118 g, 1.05 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (5 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.97 g, 1.1 mL, 3.22 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.5 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 33 as white solid (2.1 g, 86%).
1H NMR (400 MHz, C6D6): δ 7.72 (m, 2H), 7.56 (m, 3H), 7.21 (m, 2H), 7-7.1 (m, 3H), 6.8 (m, 3H), 5.4 (bs, 1H), 4.94 (bs, 1H), 4.56 (m, 1H), 3.54 (m, 3H), 3.42 (m, 1H), 3.2-3.38 (m, 9H), 3.1 (m, 2H), 2.94 (m, 1H), 2.78 (m, 1H), 2.68 (m, 2H), 2.4-2.6 (m, 3H), 2.22 (m, 1H), 2-2.12 (m, 8H), 0.9-1.9 (m, 63H), 0.66 (s, 3H).
31P NMR (161.82 MHz, C6D6): δ 145.48, 145.33 (NO rotamers observed after removing amide bond)
13C NMR (100 MHz, C6D6): δ 159.07, 155.86, 146.20, 140.19, 140.19, 137.83, 136.95, 130.65, 129.27, 128.77, 128.51, 127.55, 126.93, 125.64, 126.66, 117.5, 113.5, 86.51, 74.63, 72.62, 72.44, 67.37, 63.39, 58.64, 58.46, 56.90, 56.46, 54.72, 50.25, 44.84, 44.72, 48.38, 43.41, 43.26, 43.29, 42.55, 40.10, 39.0, 39.38, 37.31, 36.8, 36.63, 36.19, 32.27, 32.14, 29.41, 28.86, 28.61, 28.38, 27.60, 27.01, 24.74, 24.67, 24.62, 24.56, 24.51, 24.32, 24.07, 24.01, 23.00, 22.74, 21.37, 21.33, 20.03, 20.0, 19.97, 19.47, 19.01, 12.05.
Synthesis of Solid Support with Immobilized Cholesterol (N-alkyl Linkage) (35)
Succinic acid mono-{5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(10,13-dimethyl-17-octyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino)-hexyl]-pyrrolidin-3-yl} Ester (34)
Referring to scheme 11, Compound 33 (1 g, 1.07 mmol) was mixed with succinic anhydride (0.16 g, 1.61 mmol) and DMAP (0.012 g, 0.1 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (10 mL), triethylamine (0.328 g, 0.45 mL, 3.25 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (50 mL) and washed water (2×25 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The product 34 was used as such for next step without further purification (1.2 g, Quantitative).
1H NMR (400 MHz, CDCl3): δ 7.32-7.36 (m, 2H), 7.2-7.28 (m, 7H), 6.76-6.8 (m, 4H), 5.4 (bs, 1H), 4.46 (m, 2H), 3.78 (s, 6H), 3.42 (m, 1H), 3-3.18 (m, 3H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.78-2.02 (m, 7H), 0.8-1.6 (m, 42H), 0.66 (s, 3H)
13C NMR (100 MHz, CDCl3): δ 158.78, 158.62, 145.16, 139.8, 136.39, 136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91, 113.28, 56.88, 56.32, 55.45, 55.4, 50.19, 45.47, 42.51, 39.93, 39.72, 38.67, 37.14, 36.74, 36.38, 36.0, 32.1, 32.06, 28.44, 28.22, 24.5, 24.0, 23.04, 22.77, 21.24, 19.55, 18.92, 12.07, 8.72
Solid Support with Immobilized Cholesterol (N-alkyl Linkage) (3)
Succinate 34 (1.2 g, 1.16 mmol) was dissolved in dichloroethane (5 mL). To that solution DMAP (0.142 g, 1.16 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.347 g, 1.16 mmol) in acetonitrile/dichloroethane (3:1, 5 mL) was added successively. To the resulting solution triphenylphosphine (0.304 g, 1.15 mmol) in acetonitrile (2.5 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (6 g, 900 μmoles, 155 μm/g) was added. The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG 35 was measured by taking UV measurement. (63 μM/g).
Synthesis of hydroxy-prolinol-phthalimido Phosphoramidite (45)
Compound 37:
Referring to scheme 12, compound 36 (15 g, 60 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (200 mL). To this solution dimethylamino pyridine (0.733 g, 6 mmol) and DMT-Cl (21.2 g, 62.6 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The excess DMT-Cl was quenched by the addition of methanol (50 mL). The solution was dried under reduced pressure. To the residue was suspended in ethyl acetate (500 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. Upon purification over silica gel using 3% MeOH/DCM compound 37 (33 g, 77%) was obtained as white foamy solid.
1H NMR (400 MHz, DMSO-d6): δ7.22-7.38 (m, 8H), 7.16-7.2 9m, 5H), 7.06 (m, 1H), 6.84 (m, 4H), 5.34 (bs, 1H), 4.88-4.96 (m, 2H), 4.25 (m, 1H), 4 (bs, 1H), 3.7 (s, 6H), 3.4 (m, 2H), 3.04 (m, 2H), 1.86 (m, 2H).
13C NMR (10 MHz, DMSO-d6): δ 158.0, 154.28, 154.22, 149.62, 145.04, 137.15, 136.64, 135.74, 136.67, 129.58, 129.53, 128.4, 128.26, 127.81, 127.73, 127.55, 127.29, 126.65, 126.65, 123.91, 113.12, 85.24, 85.14, 68.45, 67.83, 65.96, 65.64, 64.39, 63.4, 54.99, 37.67, 36.68.
5-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol (38)
Compound 37 (8.25 g, 14.9 mmol) was dissolved in methanol (20 mL) and purged with nitrogen. To the solution were added ammonium formate (14.1 g, 223 mmol) and 10% Pd/C (0.825 g). The suspension was stirred at room temperature for 1 h. The reaction mixture was filtered through a pad of Celite and washed with methanol. The solution was concentrated to dryness under vacuum. The residue was dissolved in ethyl acetate 9250 mL) and washed with water (2×25 mL). The organic layer was dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. Product 38 (6.25 g, 98%) was used without purification for the next step.
1H NMR (400 MHz, DMSO-d6): δ 8.28 (bs, 1H), 7.36 (m, 2H), 7.18-7.3 (m, 7H), 6.84 (d, 4H), 4.2 (m, 1H), 3.7 (s, 6H), 3.6 (m, 1H), 3.02 (m, 3H), 2.8 (d, 1H), 1.74 (dd, 1H), 1.48 (m, 1H).
13C NMR (10 MHz, DMSO-d6): δ 165.02, 158.06, 149.9, 135.5, 129.74, 127.81, 127.72, 126.67, 113.15, 85.58, 69.6, 59.76, 56.81, 55.02, 53.6237.66, 14.08.
6-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-hexanoic Acid (41a)
6-amino hexanoic acid 3a) (13.1 g, 100 mmol) and phthalic anhydride 40) (14.8 g, 100 mmol) were mixed in toluene (150 mL). To the suspension was added triethyl amine (13 mL). The suspension was refluxed using Dean-stark for 16 h. When collection of water ceased, the reaction was cooled and evaporated to dryness. The residue was suspended in water and conc. hydrochloric acid (1.5 mL) was added. The suspension was stirred for 30 mins and filtered. The precipitate was washed with water and dried over sodium sulfate to afford compound 41a (24.5 g, 93%) which was used as such for the next step.
6-(1,3-Dioxo-1,3-dihydro-isoindol-2-yl)-hexanoic Acid Pentafluorophenyl Ester (43a)
Referring to scheme 12, compound 41a (13.3 g, 51 mmol) was dissolved in anhydrous dichloromethane (40 mL) and cooled to 0° C. under argon. To the solution were added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50 mmol) and pentafluoro phenol (42, 9.2 g, 50 mmol). After overnight the reaction mixture was evaporated to dryness. To the residue ethyl acetate (100 mL) was added and the filtered to remove diisopropyl urea. The precipitate was washed with ethyl acetate (50 mL). The combined organic layer was washed with saturated sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. Compound 43a (Rf=0.8 in 10% EtOAc/Hexane, 21.65 g, 92%) was obtained, which was directly used for the next step without further purification.
1H NMR (400 MHz, CDCl3): δ 7.82 (m, 2H), 7.7 (m, 2H), 3.7 (t, 2H), 2.65 (t, 2H), 1.7-1.85 (m, 4H), 1.48 (m, 2H).
13C NMR (100 MHz, CDCl3): δ 169.46, 168.62, 142.5, 140.84, 139.29, 138.32, 136.75, 134.13, 132.28, 123.39, 37.78, 33.3, 28.33, 26.21, 24.46.
2-(6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-isoindole-1,3-dione (44a)
Amine 38 (9.32 g, 22.2 mmol) and triethyl amine (4.55 g, 6.27 mL, 45 mmol) weres dissolved in anhydrous dichloromethane (20 mL) and cooled to 0° C. under argon. To that solution was added compound 43a (9.5 g, 22.2 mmol) at 0° C. The reaction mixture was brought to ambient temperature and stirred further. After 30 mins, disappearance of starting materials were ascertained by TLC. (10% MeOH/CHCl3). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaOH solution (3×50 mL) followed by water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. Upon purification over silica gel compound 44a was obtained as foamy white solid in good yield. (13.2 g, 89%).
1H NMR (400 MHz, DMSO-d6): δ 7.84 (m, 4H), 7.3 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (d, —OH), 4.38 (m, 1H), 4.1 (m, 1H), 3.72 (d, 6H), 3.55 (m, 3H), 3.3 (m, 2H), 3.12 (m, 1H), 2.97 (m, 1H), 2.2 (t, 2H), 2.0 (m, 1H), 1.9 (m, 1H), 1.82 (m, 1H), 1.44-1.6 (m, 1H), 1.3 (m, 2H), 1.14 (m, 1H),
13C NMR (100 MHz, DMSO-d6): δ 172.78, 172.11, 168.74, 168.67, 158.74, 158.55, 158.54, 145.24, 144.72, 136.46, 136.27, 137.87, 135.84, 134.18, 134.13, 134.09, 132.3, 132.27, 130.18, 130.11, 129.33, 128.22, 128.20, 128.08, 128.03, 127.93, 127.12, 126.89, 123.41, 123.38, 113.35, 113.2, 86.7, 86.06, 70.7, 69.46, 65.51, 63.67, 56.61, 56.0, 55.9, 55.42, 55.36, 54.2, 38.44, 38.0, 37.98, 36.9, 35.0, 33.4, 28.6, 28.5, 28.4, 26.79, 26.71, 25.0, 24.6, 24.5.
4-Hydroxy-prolinol-phthalimido Phosphoramidite (45a)
Compound 44a (9.0 g, 13.57 mmol) was coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.766 g, 6.8 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (20 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (6.13 g, 6.7 mL, 20.35 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC (Rf=0.7 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford compound 45a as white solid (10.5 g, 89%).
1H NMR (400 MHz, C6D6): δ 7.62 (m, 2H), 7.42 (m, 6H), 7.22 (t, 2H), 7.08 (m, 1H), 6.88 (dd, 2H), 6.78 (m, 4H), 4.66 (m, 1H), 4.56 (m, 1H), 3.72 (m, 1H), 3.5 (m, 5H), 3.3 (m, 7H), 3.22 (m, 1H), 2.1 (m, 5H), 1.74 (m, 4H), 1.56 (m, 2H), 1.26 (m, 2H), 1.1 (m, 13H).
31P NMR (161.82 MHz, C6D6): δ 145.98, 145.8, 145.63, 146.3 (Rotamers observed after due to amide bond at the ring)
13C NMR (100 MHz, C6D6): δ 171.03, 170.08, 167.98, 159.23, 159.0, 146.1, 136.76, 136.69, 136.64, 136.27, 133.35, 132.7, 130.59, 130.54, 130.46, 128.65, 128.56, 127.55, 126.97, 128.24, 128.0, 127.7, 122.84, 113.62, 113.53, 113.51, 86.57, 86.51, 72.67, 72.5, 72.33, 64.48, 58.59, 58.46, 58.41, 58.28, 57.77, 56.03, 55.97, 54.81, 54.73, 43.47, 43.35, 37.87, 36.42, 36.32, 34.94, 34.88, 33.37, 28.77, 26.94, 24.67, 24.6, 24.51, 20.10, 20.04, 19.98.
Compound 45b:
The phosphoramidite 45b is obtained from 39b in four steps as described for the synthesis of compound 45a from 39a.
Synthesis of Solid Support Immobilized with Phthalimido Group (46a)
Referring to scheme 13, Compound 44a (3 g, 4.5 mmol) was mixed with succinic anhydride (0.675 g, 6.75 mmol) and DMAP (0.055 g, 0.45 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (10 mL), triethylamine (1.37 g, 1.8 mL, 13.5 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (150 mL) and washed with 5% ice-cold citric acid (2×50 mL) followed by water (2×50 mL) and brine. The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The succinate was obtained after purification over silica gel (3.2 g, 93%).
1H NMR (400 MHz, DMSO-d6): δ 8.08 (m, 1H), 7.82 (m, 3H), 7.28 (m, 4H), 7.16 (m, 5H), 6.84 (m, 4H), 5.32 (m, 1H), 4.18 (m, 1H), 3.7 (s, 6H), 3.53 (m, 3H), 3.32 (m, 2H), 3.2 (m, 1H), 3.0 (m, 1H), 2.94 (s, 2H), 2.4 (m, 6H), 2.2 (m, 3H), 2.0 (m, 1H), 1.5 (m, 4H), 1.28 (m, 2H), 1.16 (m, 1H).
13C NMR (100 MHz, DMSO-d6): δ 172.95, 171.95, 168.78, 168.64, 158.77, 158.59, 145.18, 144.67, 136.44, 136.23, 135.8, 134.13, 133.33, 132.29, 130.21, 130.13, 128.25, 128.13, 127.98, 126.94, 123.45, 113.40, 113.26, 106.61, 86.11, 73.59, 63.67, 55.76, 55.39, 53.31, 39.64, 38.03, 35.1, 35.51, 28.56, 26.82, 24.45.
The succinate (2.7 g, 3.5 mmol) was dissolved in dichloroethane (15 mL). To that solution DMAP (0.0427 g, 3.5 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (1.086 g, 3.5 mmol) in acetonitrile/dichloroethane (3:1, 15 mL) was added successively. To the resulting solution triphenylphosphine (0.918 g, 3.5 mmol) in acetonitrile (7 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (10.5 g, 1620 μmoles, 155 μm/g) was added. The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG 46a was measured by taking UV measurement. (63 μM/g).
Synthesis of Solid Support Immobilized with Phthalimido Group (46)
The desired compound 46b is obtained from compound 44b in two steps as described for the preparation of compound 46a from the corresponding precursor 44a.
Serinol as a linker:
Synthesis of Solid Support Immobilized with Cholesterol—Serinol Linker (54)
ε-N-cholesteryloxycarbonylaminocaproic Acid (49)
Referring to Scheme 14, ε-aminocaproic acid (3.93 g, 30 mmol) was suspended in pyridine (60 mL). The flask was flushed with nitrogen and to the mixture was added N,O-bis(trimethylsilyl)acetamide (10 mL, 70 mmol) under stirring. The reaction mixture was stirred at room temperature for 30 min. Then cooled in ice bath. Cholesteryl chloroformate (13.5 g, 30 mmol) was added into reaction mixture in two portions over 2 h. The reaction was continued by stirring at room temperature for another 4 h. 2% HCl aqueous solution (150 ml) was added under cooling with ice bath. The mixture was stirred for 5 min. and then poured into a separating funnel. The product was extracted with dichloromethane (3×150 mL). The combined organic layer was washed with 2% HCl solution (2×150 mL) and with brine (2×150 mL), dried over anhydrous sodium sulfate, filtered and evaporated to dryness giving a yellow foam (14.44 g, 87%)
1H NMR (400 MHz, CDCl3): δ 5.36 (m, 1H), 4.48 (m, 1H), 3.15 (m, 2H), 2.38 (t, 2H), 1.8-2.04 (m, 5H), 1.32-1.7 (m, 19H), 0.88-1.2 (m, 22H), 0.67 (s, 3H)
13C NMR (100 MHz, CDCl3): δ 179.17, 156.42, 139.94, 126.64, 74.43, 56.82, 56.28, 50.14, 42.45, 39.88, 39.67, 38.69, 37.13, 36.69, 36.34, 35.97, 34.08, 32.05, 32.01, 29.78, 28.48, 28.30, 28.17, 26.32, 24.47, 24.44, 24.0, 23.0, 22.73, 21.19, 19.5, 18.87, 12.01.
ε-N-pentalfluorophenyl Cholesteryloxycarbonylamino Caproate (50)
Referring to scheme 14, ε-N-cholesteryloxycarbonylaminocaproic acid (49) (22.71 g, 41.9 mmol) was dissolved in anhydrous dichloromethane (40 mL) and cooled to 0° C. To the solution were added diisopropyl carbodiimide (5.17 g, 6.4 mL, 41 mmol) and triethylamine (10.2 g, 13.7 mL, 100 mmol). After stirring for 20 mins at 0° C., pentafluorophenol (7.71 g, 41.9 mmol) was added and the stirring was continued at room temperature under argon for over night. The reaction mixture was evaporated to dryness. To the residue ethyl acetate (100 mL) was added and the filtered to remove diisopropyl urea. The precipitate was washed with ethyl acetate (50 mL). The combined organic layer was washed with saturated sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. Compound 50 (Rf=0.7 in 10% EtOAc/hexane, 25.4 g, 86%) was obtained, which was directly used for the next step without further purification.
1H NMR (400 MHz, CDCl3): δ 5.38 (m, 1H), 4.47 (m, 1H), 3.2 (m, 2H), 2.36 (t, 2H), 1.81-2.05 (m, 5H), 1.3-1.7 (m, 19H), 0.89-1.21 (m, 22H), 0.68 (s, 3H)
13C NMR (100 MHz, CDCl3): δ 179.2, 156.2, 139.84, 139.29, 138.32, 136.75, 134.13, 132.28, 126.64, 123.39, 74.43, 56.82, 56.28, 50.14, 42.45, 39.88, 39.67, 38.69, 37.13, 36.69, 36.34, 35.97, 34.08, 32.05, 32.01, 29.78, 28.48, 28.30, 28.17, 26.32, 24.47, 24.44, 24.0, 23.0, 22.73, 21.19, 19.5, 18.87, 12.01.
Synthesis of [5-(2-Hydroxy-1-hydroxymethyl-ethylcarbamoyl)-pentyl]-carbamic Acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester (52)
Serinol (51) (1.37 g, 15 mmol) and triethyl amine (3.03 g, 4.15 mL, 30 mmol) were dissolved in anhydrous dichloromethane (20 mL) and cooled to 0° C. under argon. To that solution was added compound 50 (7.1 g, 10 mmol) at 0° C. The reaction mixture was brought to ambient temperature and stirred further. After 3 h, disappearance of starting materials were ascertained by TLC. (10% MeOH/CHCl3). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaOH solution (3×50 mL) followed by water and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated to dryness. Upon purification over silica gel using 5% MeOH/DCM, compound 52 was obtained as foamy white solid in good yield. (5.61 g, 90%).
1H NMR (400 MHz, DMSO-d6): δ 5.32 (m, 1H), 4.58 (t, 2H), 4.28 (m, 2H), 3.58 (m, 1H), 3.38 (m, 4H), 2.91 (t, 2H), 2.2 (m, 2H), 2.06 (t, 2H), 1.72-1.98 (m, 5H), 0.82-1.58 (m, 37H), 0.74 (s, 3H).
M/S(m/z): Calculated: 616.48 Observed: 617.5 (M++1), 636.4 (M++Na).
Synthesis of (5-{1-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-2-hydroxy-ethylcarbamoyl}-pentyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester (54)
Diol 52 (5.6 g, 9.1 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (10 mL). To this solution dimethylamino pyridine (0.110 g, 0.91 mmol) and DMT-Cl (3.23 g, 9.53 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. Due to the presence of two primary hydroxyl groups, the reaction never went to completion. The solution was dried under reduced pressure and co-evaporated with toluene to remove residual pyridine. To the residue was suspended in ethyl acetate (200 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was obtained after removal of the solvent. Upon purification over silica gel using 2% MeOH/DCM compound 53 (0.680 g, 10%) was obtained as white foamy solid.
1H NMR (400 MHz, DMSO-d6): δ 7.62 (d, 1H), 7.36 (m, 2H), 7.18-7.3 (m, 6H), 7.1 (m, 1H), 6.86 (m, 4H), 5.32 (bs, 1H), 4.6 (t, 1H), 4.28 (m, 1H), 3.98 (m, 1H), 3.72 (s, 6H), 3.42 (m, 2H), 2.98 (m, 1H), 2.9 (m, 3H), 1.72-2.3 (m, 9H), 0.8-1.58 (m, 39H), 0.64 (s, 3H).
Synthesis of Succinic Acid mono-(3-[bis-(4-methoxy-phenyl)-phenyl-methoxy]-2-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,47,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoylamino}-propyl) Ester (54)
DMT-alcohol 53 (0.650 g, 0.707 mmol) was mixed with succinic anhydride (0.100 g, 1 mmol) and DMAP (0.0123 g, 0.1 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.203 g, 0.27 mL, 2 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (20 mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) and water (2×25 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 54 as white solid (0.54 g, 78% yield; Rf=0.5 in 10% MeOH/CHCl3).
1H NMR (400 MHz, CDCl3): δ 7.26-7.32 (m, 5H), 7.16-7.18 (m, 4H), 6.84 (m, 4H), 5.38 (bs, 1H), 4.6 (t, 1H), 4.2-4.6 (m, 4H), 3.8 (s, 6H), 3.62 (m, 2H), 3.18 (m, 4H), 2.6-2.72 (m, 4H), 2.2-2.38 (m, 3H), 1.82-2.04 (m, 9H), 0.84-1.62 (m, 39H), 0.66 (s, 3H).
Synthesis of Cholesterol Immobilized on Solid Support with Serinol Linker (55)
Succinate 54 (0.51 g, 0.5 mmol) was dissolved in dichloroethane (2 mL). To that solution DMAP (0.061 g, 0.5 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.155 g, 0.5 mmol) in acetonitrile/dichloroethane (3:1, 2 mL) was added successively. To the resulting solution triphenylphosphine (0.131 g, 0.5 mmol) in acetonitrile (1 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (2.2 g, 115 μm/g) was added. The suspension was agitated for 3 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (35 μM/g).
Serinol-cholesterol-phosphoramidite (56)
Referring to Scheme 15, Compound 53 (0.92 g, 1 mmol) is coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.056 g, 0.5 mmol) is added and the mixture is dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) is added. The reaction mixture is stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC. The reaction mixture is diluted with dichloromethane (25 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue is purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford amidite 56.
Synthesis of Pyrrolidine-Cholesterol Phosphoramidite
Synthesis of 3-(Ethoxycarbonylmethyl-amino)-propionic Acid Ethyl Ester (58)
Referring to scheme 16, a 4.7M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until the completion of reaction was ascertained by TLC (19 h). After 19 h which it was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford 58 (28.8 g, 61%).
1H NMR (CDCl3, 400 MHz): δ 4.1-4.2 (m, 4H), 3.4 (s, 2H), 2.8 (t, J=6.7 Hz, 2H), 2.4 (t, J=6.7 Hz, 2H), 1.25 (m, 6H).
Synthesis of 3-[(6-Benzyloxycarbonylamino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic Acid Ethyl Ester (59)
6-benzyloxyamino hexanoic acid (13.25 g, 50 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added diisopropyl carbodiimide (6.31 g, 7.7 mL, 50 mmol) and triethylamine (10.2 g, 13.7 mL, 100 mmol). After stirring for 20 mins at 0° C., compound 58 (10.16 g, 50 mmol) was added and the stirring was continued at room temperature under argon for over night. The reaction mixture was evaporated to dryness. To the residue ethyl acetate (100 mL) was added and the filtered to remove diisopropyl urea. The precipitate was washed with ethyl acetate (50 mL). The combined organic layer was washed with 2N HCl, saturated sodium bicarbonate and water. The organic layer was dried over sodium sulfate, filtered and evaporated to dryness. Compound 59 (Rf=0.5 in 25% EtOAc/Hexane, 20.5 g) was obtained, which was directly used for the next step without further purification.
1H NMR (CDCl3, 400 MHz): δ 7.36 (m, 5H), 5.1 (s, 2H), 4.06-4.22 (m, 6H), 3.6-3.7 (m, 2H), 3.2 (m, 2H), 2.6 (m, 2H), 2.42 (m, 2H), 2.14 (m, 2H), 1.2-1.7 (m, 12H).
Synthesis of 1-(6-Benzyloxycarbonylamino-hexanoyl)-4-oxo-pyrrolidine-3-carboxylic Acid Ethyl Ester (60)
To a suspension of potassium t-butoxide (7.12 g, 64 mmol) in toluene (150 mL) at 0° C. under nitrogen, was added diester 59 (20 g, 44 mmol) in toluene (25 mL) over a 10 min period. The solution was stirred for 30 min at 0° C. and 5 mL of glacial acetic acid was added, immediately followed by 25 g of NaH2PO4.H2O in 250 mL of ice-cold water. The resultant mixture was extracted with chloroform (3×200 mL), and the combined organic extracts were washed twice with phosphate buffer (2×25 mL, pH=7.0), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was dissolved in toluene (300 mL), cooled to 0° C., and extracted with cold pH 9.5 carbonate buffer (3×150 mL). The aqueous extracts were converted to pH 3 with phosphoric acid, and extracted with chloroform (5×125 mL) which were combined, dried, and evaporated to a afford keto ester 60 (12 g, 45%).
The toluene fraction was washed with water (25 mL), dried and evaporated to afford ketoester 61 (7.6 g, 28%).
1H NMR (CDCl3, 400 MHz): δ 7.35 (m, 5H), 5.1 (s, 2H), 4.05-4.34 (m, 6H), 3.8 (m, 1H), 3.2 (m, 4H), 2.6 (m, 1H), 2.2-2.4 (m, 1H), 1.68 (m, 1H), 1.52 (m, 1H), 1.24-1.4 (m, 6H).
Synthesis of 1-(6-Benzyloxycarbonylamino-hexanoyl)-4-hydroxy-pyrrolidine-3-carboxylic Acid Ethyl Ester (62)
To a solution of sucrose (3 g) in distilled water (40 mL) was added Baker's yeast (2 g). The suspension was heated at 32° C. for 1 h. The content of the flask was then poured into a flask containing ketoester 60 (4 g, 9.88 mmol, dissolved in 4 mL of methanol). Stirring was continued at 32° C. for 24 h after which additional sucrose (3 g) in warm (40° C.) distilled water was added. After 48 h, the suspension was filtered through a pad of Celite. The pad was washed with water and the aqueous layer was extracted with ethyl acetate (3×250 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to flash chromatography (30% EtOAc/Hexane) to afford alcohol 62 (1.7 g, 42%).
1H NMR (CDCl3, 400 MHz): δ 7.32 (m, 5H), 5.12 (s, 2H), 4.56 (m, 1H), 4.2 (m, 2H), 3.9 (m, 1H), 3.83 (m, 1H), 3.63 (m, 1H), 3.48 (m, 1H), 2.82-3.06 (m, 3H), 2.2 (t, 2H), 1.22-1.41 (m, 9H). (Also observed minor rotamer due to amide bond)
13C NMR (CDCl3, 100 MHz): δ 173.2, 172.5, 171.41, 156.78, 136.74, 128.60, 128.17, 128.11, 70.19, 68.38, 66.56, 60.56, 58.22, 57.71, 55.36, 54.60, 52.36, 40.78, 37.73, 34.2, 29.64, 24.22, 21.66, 14.29.
Synthesis of [6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic Acid Benzyl Ester (63)
To the solution of lithium borohydride (0.305 g, 13 mmol) in anhydrous tetrahydrofuran (25 mL) was added a solution of ethyl ester 62 (3.74 g, 9.2 mmol) in THF (25 mL) over a period of 30 mins at 0° C. After the addition the reaction mixture was brought to room temperature and stirred further under argon. The completion of the reaction was ascertained by TLC after 4 h. (Rf=0.4 in 10% MeOH/CHCl3). The reaction mixture was evaporated to dryness and cooled to 0° C. To the residue 3N HCl (40 mL) was added slowly. After stirring for 30 mins the product was extracted with dichloromethane (3×75 mL). The combined organic layer was washed with brine and dried over sodium sulfate. Organic layer was filtered and evaporated to dryness. Compound 63 was purified by column chromatography first by eluting with dichloromethane/methanol (5%) (3.2 g, 92%).
1H NMR (CDCl3, 400 MHz): δ 7.34 (m, 5H), 5.16 (s, 2H), 4.64 (m, 1H), 4.4 (bs, 1H), 4.2 (m, 1H), 3.78 (m, 2H), 3.62 (m, 3H), 3.5 (m, 2H), 2.06 (m, 4H), 1.55 (m, 4H), 1.2 (m, 2H).
Synthesis of (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic Acid Benzyl Ester (64)
Referring to scheme 16, compound 63 (3.65 g, 10 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (10 mL). To this solution dimethylamino pyridine (0.122 g, 1 mmol) and DMT-Cl (3.55 g, 10.5 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The excess DMT-Cl was quenched by the addition of methanol (10 mL). The solution was dried under reduced pressure. To the residue was suspended in ethyl acetate (200 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was obtained after removal of the solvent. Upon purification over silica gel using 3% MeOH/DCM compound 64 (5.9 g, 88%) was obtained as white foamy solid.
1H NMR (CDCl3, 400 MHz): δ 7.24-7.38 (m, 13H), 7.18 (m, 2H), 6.84 (m, 3H), 5.1 (s, 2H), 4.96 (m, 1H), 4.36 (m, 2H), 3.74-3.8 (m, 8H), 3.52 (m, 2H), 3.2 (m, 3H), 1.88-2.38 (m, 4H), 1.28-1.72 (m, 6H)
13C NMR (100 MHz, CDCl3): δ 174.7, 172.7, 171.9, 171.3, 171.2, 158.8, 158.7, 158.6, 158.5, 158.4, 158.3, 156.7, 156.7, 156.6, 147.5, 145.8, 145.2, 144.9, 144.7, 144.4, 139.6, 137.1, 137.04, 137.01, 136.9, 136.82, 136.78, 136.55, 136.47, 136.45, 136.3, 136.28, 135.93, 135.85, 135.81, 130.2, 130.1, 130.0, 129.9, 129.3, 128.69, 128.66, 128.22, 128.16, 128.0, 127.99, 127.94, 127.91, 127.77, 113.52, 113.43, 113.35, 113.3, 113.24, 113.19, 113.03, 86.8, 86.1, 85.9, 73.0, 71.6, 71.5, 70.5, 69.3, 67.3, 67.1, 68.76, 68.71, 64.38, 63.7, 60.58, 60.0, 56.4, 55.8, 55.7, 55.45, 55.41, 55.35, 55.33, 40.97, 40.87, 40.77, 37.13, 36.83, 35.13, 35.00, 34.81, 34.6, 33.3, 29.8, 26.73, 25.5, 26.4, 26.2, 24.9, 24.6, 24.5, 24.3, 24.2, 21.1, 14.3.
Synthesis of 6-Amino-1-{3-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-hexan-1-one (65)
Compound 64 (5.9 g, 8.84 mmol) was dissolved in methanol (10 mL) and purged with argon. To the solution was added 10% palladium on carbon (0.6 g). The flask was purged with hydrogen 2 times and stirred further at room temperature under hydrogen atmosphere for overnight. The disappearance of the starting material was confirmed by the TLC. The reaction mixture was filtered through a pad of Celite and washed with methanol. The combined organic layer was concentrated under reduced pressure to afford compound 65 (4.3 g, 92%) as white solid. This was used as such for the next step.
1H NMR (DMSO-d6, 400 MHz): δ 7.16-7.36 (m, 9H), 6.88 (m, 4H), 4.4 (m, 1H), 4.16 (m, 1H), 3.72 (m, 6H), 3.56 (dd, 1H), 3.34 (m, 1H), 3.14 (m, 1H), 3.0 (m, 1H), 2.7 (m, 2H), 2.2 (m, 2H), 1.8-2.1 (m, 3H), 1.28-1.58 (m, 6H), 1.16 (m, 2H).
13C NMR (DMSO-d6, 100 MHz): δ 170.84 (Minor disappears at 80° C.), 170.75, 165.82, 158.1, 157.98, 145.1, 144.76, 135.86, 135.74, 129.61, 129.57, 127.91, 127.81, 127.57, 126.61, 113.23, 113.31, 85.79, 85.11, 68.55 63.33, 56.76, 55.07, 55.02, 38.63, 36.27, 33.89, 32.34, 27.12, 27.05, 23.91, 20.77, 14.09.
Synthesis of (6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester (66)
Referring to scheme 16, compound 65 (7.75 g, 14.5 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added triethylamine (3 g, 4.2 mL, 30 mmol) and cholesteryl chloroformate (6.5 g, 29 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 2h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was evaporated under the vacuum to afford the crude product. Compound 66 (12.4 g, 88%) was obtained as a white foamy solid after column chromatography over silica gel using 3% MeOH/DCM.
1H NMR (400 MHz, DMSO-d6): δ 7.12-7.3 (m, 8H), 6.95 (m, 1H), 6.84 (m, 4H), 5.3 (bs, 1H), 4.92 and 4.84 (d, OH, exchangeable with D2O), 4.21-4.38 (m, 2H), 4.35 (m, 1H), 3.7 (s, 6H), 3.54 (m, 1H), 3.28 (m, 2H), 3.12 (m, 1H), 2.84-2.98 (m, 3H), 2.12-2.28 (m, 3H), 1.7-2.0 (m, 7H), 0.8-1.52 (m, 40H), 0.6 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 170.8, 158.0, 157.9, 155.6, 145.0, 139.7, 135.8, 135.7, 129.5, 127.7, 127.5, 121.7, 113.1, 113.0, 85.7, 85.1, 72.7, 68.5, 63.3, 56.1, 55.5, 54.9, 49.4, 41.8, 36.5, 35.2, 31.3, 27.7, 27.3, 26.0, 24.1, 23.8, 23.2, 22.6, 22.3, 20.5, 18.9, 18.5, 11.6.
M/S (ESI): Calculated: 944.63 Observed: 967.6 (M++Na).
Synthesis of pyrrolidine-cholesterol Phosphoramidite (67)
Compound 66 (0.15 g, 0.158 mmol) was coevaporated with toluene (5 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.0089 g, 0.079 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in the mixture of anhydrous acetonitrile/dichloromethane (2:1, 1 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.0714 g, 0.0781 mL, 0.237 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC (1:1 ethyl acetate:hexane). The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (10 mL) and washed with 5% NaHCO3 (4 mL) and brine (4 mL). The ethyl acetate layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The resulting mixture was chromatographed (50:49:1, EtOAc:Hexane:triethlyamine) to afford 67 as white foam (0.152 g, 84%).
1H NMR (CDCl3, 400 MHz): δ 7.36 (m, 2H), 7.24 (m, 7H), 6.8 (m, 4H), 5.38 (m, 1H), 4.7 (m, 1H), 4.5 (m, 1H), 4.36 (m, 1H), 3.5-3.8 (m, 9H), 3.36-3.6 (m, 4H), 3.14 (m, 3H), 2.58 (m, 2H), 1.8-2.38 (m, 12H), 0.84-1.68 (m, 51H), 0.66 (s, 3H).
31P NMR (161.82 MHz, CDCl3): δ 146.3, 146.2, 145.98, 145.8, 145.63, 145.4 (multiple peaks due mixer of diastereomer and Rotamers observed after due to amide bond at the ring)
13C NMR (CDCl3, 100 MHz): δ 171.6, 158.75, 158.58, 156.36, 145.32, 144.78, 140.10, 136.48, 136.36, 136.32, 135.84, 130.19, 129.24, 128.44, 128.27, 128.21, 128.13, 127.97, 127.15, 126.92, 125.51, 122.62, 117.87, 117.79, 113.40, 113.25, 86.16, 86.11, 74.31, 72.39, 63.92, 58.5, 58.3, 58.1, 56.8, 56.3, 55.9, 55.8, 55.4, 55.3, 52.2, 43.4, 43.3, 42.5, 40.8, 39.9, 39.7, 38.7, 37.2, 36.7, 36.3, 36.0, 35.0, 32.1, 32.0, 30.0, 28.45, 28.4, 28.2, 26.8, 24.8, 24.7, 24.69, 24.6, 24.5, 24.0, 23.0, 22.7, 21.6, 21.2, 20.6, 20.59, 20.52, 19.5, 18.9, 11.6
Synthesis of Succinic Acid mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) Ester (68)
Referring to scheme 17, Compound 66(12 g, 12.69 mmol) was mixed with succinic anhydride (1.9 g, 19 mmol) and DMAP (1.56 g, 13 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (50 mL), triethylamine (2 g, 3.6 mL, 26 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 68 as white solid (12.1 g, 91% yield; Rf=0.5 in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 7.12-7.32 (m, 9H), 6.82 (m, 4H), 5.3 (m, 2H), 4.26 (m, 1H), 4.06 (m, 1H), 3.6-3.78 (m, 8H), 3.52 (m, 1H), 3.2 (m, 1H), 3 (m, 2H), 2.88 (m, 2H), 2.7 (m, 1H), 2.1-2.24 (m, 6H), 1.84-2.04 (m, 3H), 1.75 (m, 4H), 0.8-1.52 (m, 39H), 0.62 (m, 3H)
13C NMR (100 MHz, DMSO-d6): δ 173.42, 171.97, 170.66, 158.12, 157.99, 156.63, 144.97, 144.67, 139.77, 135.73, 135.59, 135.38, 129.61, 127.88, 127.8, 127.57, 126.61, 121.8, 113.2. 113.12, 85.96, 85.26, 72.81, 72.73, 63.24, 56.12, 55.58, 55.0, 54.97, 54.84, 49.47, 41.85, 36.60, 35.66, 35.22, 33.09, 31.38, 29.33, 28.84, 28.74, 27.9, 27.8, 27.4, 25.96, 24.40, 23.86, 23.23, 22.66, 22.39, 20.57, 18.98, 18.53, 11.66, 10.01.
Synthesis of Cholesterol Immobilized Solid Support with Pyrrolidine Linker (69)
Succinate 68 (8.4 g, 8 mmol) was dissolved in dichloroethane (40 mL). To that solution DMAP (0.977 g, 8 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (2.49 g, 8 mmol) in acetonitrile/dichloroethane (3:1, 40 mL) was added successively. To the resulting solution triphenylphosphine (2.1 g, 8 mmol) in acetonitrile (20 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (30 g, 155 μm/g) was added. The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (82 μM/g).
Synthesis of phthalimido-pyrrolidine Phosphoramidite
Synthesis of 3-(Benzyloxycarbonyl-ethoxycarbonylmethyl-amino)-propionic Acid Ethyl Ester (70)
To a solution of diester 58 (3.88 g, 20.2 mmol) in dry acetonitrile (40 mL) at 0° C., under Argon, was added slowly benzyl chloroformate (3.17 mL, 1.1 equiv.). The solution was stirred at 0° C. for 1 h after which it was poured into water (50 mL). The phases were separated and the aqueous layer was extracted with dichloromethane (3×50 mL). the combined organic extracts were washed with 5% HCl, water, brine and dried over anhydrous sodium sulfate. Evaporation of the solvents was followed by distillation to afford compound 70 as a colorless oil (5.4 g, 80%).
1H NMR (400 MHz, CDCl3): δ 7.18-7.4 (m, 5H), 5.13 (s, 2H), 4.12 (q, 2H, J=7.1 Hz), 4.06 (q, 2H, J=7.1 Hz), 3.58 (t, 2H, J=6.4 Hz), 2.6 (t, 2H, J=6.4 Hz), 1.18 (t, 3H, J=7.1 Hz), 1.2 (t, 3H, J=7.1 Hz).
13C NMR (100 MHz, CDCl3): δ 172.7, 170.4, 156.7, 136.8, 129, 128.9, 128.4, 128.3, 128.2, 68, 61.6, 61.1, 50.9, 45.8, 34.6, 14.6.
Synthesis of 4-Oxo-pyrrolidine-1,3-dicarboxylic acid 1-benzyl ester 3-ethyl Ester (71)
To a suspension of potassium t-butoxide (2.52 g, 22.4 mmol, 1.4 equiv.) in toluene (50 mL) at 0° C. under nitrogen, was added diester 70 (5.41 g, 16 mmol) in toluene (10 mL) over a 10 min period. The solution was stirred for 30 min at 0° C. and 2 mL of glacial acetic acid was added, immediately followed by 10 g of NaH2PO4.H2O in 100 mL of ice-cold water. The resultant mixture was extracted with chloroform (3×150 mL), and the combined organic extracts were washed twice with phosphate buffer (2×25 mL, pH=7.0), dried over anhydrous sodium sulfate and evaporated to dryness. The residue was dissolved in toluene (200 mL), cooled to 0° C., and extracted with cold pH 9.5 carbonate buffer (3×150 mL). The aqueous extracts were converted to pH 3 with phosphoric acid, and extracted with chloroform (5×125 mL) which were combined, dried, and evaporated to a afford keto ester 71 (2.2 g, 42%).
The toluene fraction was washed with water (10 mL), dried and evaporated to afford ketoester 72 (1.3 g, 24%).
1H NMR (400 MHz, CDCl3): δ 7.2 (m, 5H), 5.18 (s, 2H), 4.25 (m, 4H), 4.1 (m, 1H), 3.94 (m, 1H), 3.62 (m, 1H), 1.3 (m, 3H).
13C NMR (100 MHz, CDCl3): δ 203.29, 166.14, 153.56, 136.36, 127.89, 127.34, 127.04, 65.96, 60.75, 52.77, 51.84, 45.55, 13.41
Synthesis of 4-Hydroxy-pyrrolidine-1,3-dicarboxylic acid 1-benzyl Ester 3-ethyl Ester
To a solution of sucrose (3 g) in distilled water (40 mL) was added Baker's yeast (2 g). The suspension was heated at 32° C. for 1 h. The content of the flask was then poured into a flask containing ketoester 71 (2.9 g, 9.88 mmol, dissolved in 4 mL of methanol). Stirring was continued at 32° C. for 24 h after which additional sucrose (3 g) in warm (40° C.) distilled water was added. After 48 h, the suspension was filtered through a pad of Celite. The pad was washed with water and the aqueous layer was extracted with ethyl acetate (3×250 mL). The combined organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was subjected to flash chromatography (30% EtOAc/Hexane) to afford alcohol 73 (1.2 g, 41%).
1H NMR (CDCl3, 400 MHz): δ 7.36 (m, 5H), 5.1 (m, 3H), 4.1 (m, 3H), 3.88 (m 2H), 3.5 (m, 1H), 3.34 (m, 2H), 1.2 (m, 3H) (Also observed minor rotamer due to amide bond).
Synthesis of 3-Hydroxy-4-hydroxymethyl-pyrrolidine-1-carboxylic Acid Benzyl Ester (74)
To the solution of lithium borohydride (0.305 g, 13 mmol) in anhydrous tetrahydrofuran (25 mL) was added a solution of ethyl ester 73 (2.69 g, 9.2 mmol) in THF (25 mL) over a period of 30 mins at 0° C. After the addition the reaction mixture was brought to room temperature and stirred further under argon. The completion of the reaction was ascertained by TLC after 4 h. (Rf=0.3 in 10% MeOH/CHCl3). The reaction mixture was evaporated to dryness and cooled to 0° C. To the residue 3N HCl (40 mL) was added slowly. After stirring for 30 mins the product was extracted with dichloromethane (3×75 mL). The combined organic layer was washed with brine and dried over sodium sulfate. Organic layer was filtered and evaporated to dryness. Compound 74 was purified by column chromatography first by eluting with dichloromethane/methanol (5%) (1.98 g, 85%).
1H NMR (CDCl3, 400 MHz): δ 7.36 (m, 5H), 5.16 (s, 2H), 4.62 (m, 1H), 4.4 (bs, 1H), 4.2 (m, 1H), 3.8 (m, 2H), 3.64 (m, 3H), 3.5 (m, 2H). (Also observed minor rotamer due to amide bond).
Synthesis of 3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidine-1-carboxylic Acid Benzyl Ester (75)
Referring to scheme 18, compound 74 (4.39 g, 17.5 mmol) was co-evaporated with anhydrous pyridine three times and then dissolved in pyridine (30 mL). To this solution dimethylamino pyridine (0.213 g, 1.75 mmol) and DMT-Cl (6.22 g, 18.4 mmol, 1.05 equiv.) were added at room temperature. The reaction mixture was stirred at room temperature for 16 h. The excess DMT-Cl was quenched by the addition of methanol (10 mL). The solution was dried under reduced pressure. To the residue was suspended in ethyl acetate (300 mL) and washed with saturated bicarbonate solution, brine and water. The organic layer was dried over anhydrous sodium sulfate, filtered and evaporated. The crude product was obtained after removal of the solvent. Upon purification over silica gel using 3% MeOH/DCM compound 75 (8.46 g, 87%) was obtained as white foamy solid.
1H NMR (CDCl3, 400 MHz): δ 7.18-7.4 (m, 14H), 6.8 (m, 4H), 5.1 (s, 2H), 5.0 (m, 1H), 4.54 (m, 1H), 4.18 (m, 2H), 3.78 (s, 6H), 3.6 (m, 2H), 3.14 (m, 1H), 2.02 (m, 1H), 1.74 (m, 1H). (Also observed minor rotamer due to amide bond).
13C NMR (CDCl3, 100 MHz): δ 158.81, 158.59, 147.52, 145.18, 139.65, 136.64, 136.31, 130.17, 129.33, 128.76, 128.58, 128.37, 128.28, 128.18, 128.05, 127.96, 127.28, 126.93, 113.35, 113.24, 86.1, 81.62, 69.93, 67.66, 67.16, 66.81, 55.45, 55.39, 37.64.
Synthesis of 4-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-pyrrolidin-3-ol (76)
Compound 75 (8.25 g, 14.9 mmol) was dissolved in methanol (20 mL) and purged with argon. To the solution was added 10% palladium on carbon (0.825 g). The flask was purged with hydrogen 2 times and stirred further at room temperature under hydrogen atmosphere for 3 h. The disappearance of the starting material was confirmed by the TLC. The reaction mixture was filtered through a pad of Celite and washed with methanol. The combined organic layer was concentrated under reduced pressure to afford amine 76 (6.12 g, 98%) as white solid. This was used as such for the next step.
1H NMR (DMSO-d6, 400 MHz): δ 8.3 (s, 1H), 7.38 (m, 2H), 7.22 (m, 7H), 6.84 (m, 4H), 4.2 (m, 1H), 3.7 (s, 6H), 3.6 (m, 1H), 3.0 (m, 3H), 2.8 (m, 1H), 1.74 (m, 1H), 1.5 (m, 1H).
13C NMR (DMSO-d6, 100 MHz): δ 165.02, 158.06, 144.90, 135.55, 129.74, 127.81, 127.72, 126.67, 113.15, 85.58, 69.6, 59.76, 56.81, 55.02, 53.62.
Synthesis of 2-(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-isoindole-1,3-dione (7)
Referring to scheme 18, compound 76 (4.2 g, 10 mmol) was dissolved in anhydrous dichloromethane (25 mL) and cooled to 0° C. To the solution were added triethylamine (1.01 g, 1.4 mL, 10 mmol) and ester 43 (4.3 g, 10 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 2h. The completion of the reaction was ascertained by TLC (10% MeOH/CHCl3). The reaction mixture was diluted with DCM (100 mL) and washed with 10% NaOH solution. The organic layer was washed with brine, water and dried over anhydrous sodium sulfate and filtered. The crude product was obtained by evaporating the solvent under the vacuum. Compound 77 (5.4 g, 81%) was obtained as a white foamy solid after column chromatography over silica gel using 4% MeOH/DCM.
1H NMR (DMSO-d6, 400 MHz): δ 7.84 (m, 4H), 7.28 (m, 4H), 7.18 (m, 5H), 6.86 (m, 4H), 4.98 (d, —OH, D2O exchangeable), 4.38 (m, 1H), 4.3 (m, 1H), 3.72 (s, 6H), 3.53 (m, 3H), 3.3 (m, 1H), 3.14 (m, 1H), 2.98 (m, 2H), 2.2 (m, 2H), 2.0 (m, 2H), 1.44-1.62 (m, 2H), 1.3 (m, 2H), 1.13 (m, 1H)
13C NMR (DMSO-d6, 100 MHz): δ 172.78, 172.11, 168.74, 168.67, 158.74, 158.55, 158.54, 145.24, 144.72, 136.46, 136.27, 137.87, 135.84, 134.18, 134.13, 134.09, 132.3, 132.27, 130.18, 130.11, 129.33, 128.22, 128.20, 128.08, 128.03, 127.93, 127.12, 126.89, 123.41, 123.38, 113.35, 113.2, 86.7, 86.06, 70.7, 69.46, 65.51, 63.67, 56.61, 56.0, 55.9, 55.42, 55.36, 54.2, 38.44, 38.0, 37.98, 36.9, 35.0, 33.4, 28.6, 28.5, 28.4, 26.79, 26.71, 25.0, 24.6, 24.5.
Pyrrolidine-phthalimido Phosphoramidite (78)
Compound 77 (1.5 g, 2.26 mmol) was coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.127 g, 1.13 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in acetonitrile/dichloroethane (10 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.362 g, 1.49 mL, 4.52 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.4 in 1:1 ethyl acetate:hexane). The reaction mixture was concentrated undervacuum and the residue was dissolved in ethyl acetate (100 mL). The organic layer was washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford compound 78 as white solid (1.66 g, 85%).
1H NMR (400 MHz, C6D6): δ 7.62 (m, 2H), 7.42 (m, 6H), 7.22 (t, 2H), 7.08 (m, 1H), 6.88 (dd, 2H), 6.78 (m, 4H), 4.66 (m, 1H), 4.56 (m, 1H), 3.72 (m, 1H), 3.5 (m, 5H), 3.3 (m, 7H), 3.22 (m, 1H), 2.1 (m, 5H), 1.74 (m, 4H), 1.56 (m, 2H), 1.26 (m, 2H), 1.1 (m, 13H).
31P NMR (161.82 MHz, C6D6): δ 146.3, 146.2, 145.98, 145.8, 145.63, 145.4 (multiple peaks due mixer of diastereomer and Rotamers observed after due to amide bond at the ring)
13C NMR (100 MHz, C6D6): δ 171.03, 170.08, 167.98, 159.23, 159.0, 146.1, 136.76, 136.69, 136.64, 136.27, 133.35, 132.7, 130.59, 130.54, 130.46, 128.65, 128.56, 127.55, 126.97, 128.24, 128.0, 127.7, 122.84, 113.62, 113.53, 113.51, 86.57, 86.51, 72.67, 72.5, 72.33, 64.48, 58.59, 58.46, 58.41, 58.28, 57.77, 56.03, 55.97, 54.81, 54.73, 43.47, 43.35, 37.87, 36.42, 36.32, 34.94, 34.88, 33.37, 28.77, 26.94, 24.67, 24.6, 24.51, 20.10, 20.04, 19.98.
Synthesis of Succinic Acid mono-{4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-[6-(1,3-dioxo-1,3-dihydro-isoindol-2-yl)-hexanoyl]-pyrrolidin-3-yl} Ester (79)
Referring to scheme 19, Compound 77 (2 g, 3 mmol) was mixed with succinic anhydride (0.600 g, 6 mmol) and DMAP (0.366 g, 3 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.913 g, 1.25 mL, 9 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 4 h. It was then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 50 mL) and water (2×50 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography using 6% MeOH/DCM to afford compound 79 as white solid (2.05 g, 89% yield; Rf=0.4 in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 7.8 (m, 4H), 7.26 (m, 4H), 7.14 (m, 5H), 6.83 (m, 4H), 4.92 (d, —OH, D2O exchangeable), 4.38 (m, 1H), 4.1 (m, 1H), 3.68 (s, 6H), 3.52 (m, 2H), 3.3 (m, 2H), 3.1 (m, 1H), 2.95 (m, 1H), 2.18 (m, 6H), 1.98 (m, 2H), 1.44-1.58 (m, 4H), 1.26 (m, 2H)
13C NMR (100 MHz, DMSO-d6): δ 170.78, 167.94, 158.07, 157.96, 145.07, 135.86, 135.44, 134.36, 131.61, 129.6, 127.78, 127.57, 126.58, 122.99, 113.10, 85.10, 68.56, 54.98, 37.28. 27.87, 26.01, 24.03.
Synthesis of Phthalimido-Pyrrolidine Immobilized on a Solid Support (80)
Succinate 79 (0.900 g, 1.17 mmol) was dissolved in dichloroethane:ACN (1:1, 5 mL). To that solution DMAP (0.144 g, 1.17 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.360 g, 1.17 mmol) in acetonitrile/dichloroethane (3:1, 5 mL) was added successively. To the resulting solution triphenylphosphine (0.306 g, 1.17 mmol) in acetonitrile (2.5 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (3.5 g, 155 μm/g) was added. The suspension was agitated for 4 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG 80 was measured by taking UV measurement. (87 μM/g)
Synthesis of Extended Steroid Conjugates with Hydroxyl-Prolinol Linker
Synthesis of 4-(3-Hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoic Acid Dioctadecylamide (82)
Lithocholic acid (81) (7.1 g, 18.8 mmol) was dissolved in anhydrous tetrahydrofuran (60 mL). Isobutylchloroformate (2.6 g, 2.6 mL, 18.8 mmol) was added followed by the addition of triethylamine (3.84 g, 5.3 mL, 38 mmol) and dioctadecylamine (9.8 g, 18.77 g). The reaction mixture was brought to ambient temperature and allowed to stir over night. The reaction mixture was concentrated under vacuum, and the residue was dissolved in dichloromethane (250 mL). The organic layer was washed with 5% sodium bicarbonate, 3% aqueous HCl and water. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure to afford amide 82 (15.5 g) in 93% yield. This was used as such for the next step.
1H NMR (400 MHz, CDCl3): δ 3.84 (d, —OH, D2O exchangeable), 3.64 (m, 1H), 3.16-3.34 (m, 4H), 2.32 (m, 1H), 2.18 (m, 1H), 1.22-1.98 (m, 83H), 0.84-1.18 (m, 17H), 0.64 (s, 3H)
13C NMR (100 MHz, CDCl3): δ 173.3, 72.1, 71.04, 56.7, 56.3, 56.19, 48.2, 46.07, 42.96, 42.32, 40.65, 40.41, 36.70, 36.07, 35.87, 35.57, 34.79, 32.13, 31.92, 30.77, 30.37, 29.83, 29.8, 29.78, 29.72, 29.68, 29.4, 28.47, 28.3, 28.03, 27.41, 27.29, 27.13, 27.09, 26.63, 24.45, 23.59, 22.9, 21.05, 19.41, 18.76, 18.48, 14.32, 12.28.
Synthesis of Carbonic Acid 17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-yl ester 2,5-dioxo-pyrrolidin-1-yl Ester (83)
Referring to scheme 20, amide 82 (15.5 g, 17.6 mmol) was dissolved in anhydrous dichloromethane (150 mL). To the solution were added disuccinimidyl carbonate (6.76 g, 26.4 mmol), triethylamine (10 mL) and acetonitrile (50 mL). The reaction mixture was stirred at room temperature under argon for 6h and then evaporated dryness. The residue was dissolved in dichloromethane (300 mL). It was washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Compound 83 (12.3 g, 71%) was obtained as colorless powder after drying in high vacuum, which was directly used for the next step without further purification.
1H NMR (400 MHz, CDCl3): δ 4.7 (m, 1H), 3.15-3.34 (m, 4H), 2.82 (s, 4H), 2.32 (m, 1H), 2.16 (m, 1H), 1.66-2.0 (m, 10H), 1.2-1.58 (m, 78H), 0.86-1.12 (14H), 0.64 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 173.34, 168.98, 151.08, 83.17, 71.37, 56.58, 56.31, 48.21, 46.06, 42.91, 42.09, 40.61, 40.26, 35.95, 35.81, 35.33, 34.96, 34.75, 34.69, 32.10, 31.19, 31.88, 29.89, 29.85, 29.79, 29.54, 28.41, 28.27, 27.96, 27.26, 27.09, 27.06, 26.42, 25.82, 25.77, 25.73, 25.66, 25.6, 24.38, 23.56, 23.34, 22.87, 21.02, 20.35, 19.37, 18.83, 18.73, 18.53, 14.3, 12.25.
Synthesis of (6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-yl Ester (84)
Amine 5 (4.22 g, 7.9 mmol) was dissolved in anhydrous dichloromethane (25 mL) and cooled to 0° C. To the solution were added pyridine (10 mL) and compound 83 (8.1 g, 7.9 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further for 3h. The completion of the reaction was ascertained by TLC (EtOAc, Rf=0.8). The reaction mixture was diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer was dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 12 (8.8 g, 77%) was obtained as a white solid after column chromatography over silica gel.
1H NMR (400 MHz, DMSO-d6): δ 7.2-7.38 (m, 9H), 6.76 (m, 4H), 4.0 (m, 2H), 3.72 (s, 6H), 3-3.18 (m, 3H), 2.96 (m, 2H), 2.5-2.6 (m, 3H), 2.12-2.38 (m, 6H), 1.22-1.98 (m, 89H), 0.84-1.18 (m, 23H), 0.64 (s, 3H)
13C NMR (100 MHz, DMSO-d6): δ 171.89, 171.38, 158.74, 158.56, 156.70, 156.6, 145.28, 144.77, 136.49, 136.33, 135.89, 135.8, 130.19, 130.15, 128.25, 128.20, 128.09, 127.94, 127.13, 126.90, 113.37, 113.21, 72.1, 71.04, 56.7, 56.3, 56.19, 48.2, 46.07, 42.96, 42.32, 40.65, 40.41, 36.70, 36.07, 35.87, 35.57, 34.79, 32.13, 31.92, 30.77, 30.37, 29.83, 29.8, 29.78, 29.72, 29.68, 29.4, 28.47, 28.3, 28.03, 27.41, 27.29, 27.13, 27.09, 26.63, 24.45, 23.59, 22.9, 21.05, 19.41, 18.76, 18.48, 14.32, 12.28.
Synthesis of Extended Steroid Conjugates Phosphoramidite with Hydroxyl-Prolinol Linker (85)
Compound 84 (5.8 g, 4 mmol) was coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.225 g, 2 mmol) was added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture was dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.8 g, 1.97 mL, 6 mmol) was added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction was ascertained by TLC(Rf=0.7 in 1:1 ethyl acetate:hexane). The reaction mixture was diluted with dichloromethane (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 85 as white solid (5.45 g, 83%).
1H NMR (400 MHz, C6D6): δ 7.62 (m, 2H), 7.46 (m, 4H), 7.24 (m, 2H), 7.08 (m, 1H), 6.8 (m, 4H), 4.9 (m, 1H), 4.6 (m, 2H), 3.74 (m, 1H), 3.5 (m, 3H), 3.4 (m, 2H), 3.36 (2s, 6H), 3-3.22 (m, 4H), 0.8-2.4 (m, 133H), 0.62 (s, 3H)
31P NMR (161.82 MHz, CDCl3): δ 148.26 (high in integration), 148.01, 147.6 (due to rotamer
13C NMR (100 MHz, CDCl3): δ 171.79, 171.61, 158.75, 158.58, 156.59, 145.31, 144.77, 136.47, 136.35, 136.31, 135.86, 130.22, 130.19, 128.28, 128.20, 128.11, 127.95, 127.15, 126.91, 113.39, 113.24, 86.11, 71.98, 70.81, 70.69, 72.93, 72.2, 71.98, 70.81, 70.81, 70.69, 64.37, 63.92, 58.55, 58.35, 58.36, 58.16, 59.57, 55.86, 55.44, 55.39, 46.31, 44.70, 44.65, 43.36, 43.34, 41.08, 35.08, 33.45, 32.13, 30.23, 29.92, 29.88, 29.72, 29.58, 26.32, 26.26, 24.85, 24.78, 24.68, 22.9, 20.58, 14.34.
Synthesis of Succinic Acid mono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(3-dioctadecylcarbamoyl-1-methyl-propyl)-1,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) Ester (86)
Referring to scheme 21, Compound 84 (1.44 g, 1 mmol) was mixed with succinic anhydride (0.15 g, 1.5 mmol) and DMAP (0.0122 g, 0.1 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.101 g, 0.14 mL, 1 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) and water (2×25 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 86 as white solid (1.1 g, 71% yield; Rf=0.5 in 10% MeOH/CHCl3).
1H NMR (400 MHz, DMSO-d6): δ 7.62 (m, 2H), 7.46 (m, 4H), 7.24 (m, 2H), 7.08 (m, 1H), 6.8 (m, 4H), 4.9 (m, 1H), 4.6 (m, 2H), 3.74 (m, 1H), 3.5 (m, 3H), 3.4 (m, 2H), 3.36 (2s, 6H), 2.82 (s, 4H), 2.32 (m, 1H), 2.16 (m, 1H), 1.66-2.0 (m, 10H), 1.2-1.58 (m, 78H), 0.86-1.12 (14H), 0.64 (s, 3H).
13C NMR (100 MHz, DMSO-d6): δ 176.59, 172.22, 158.78, 158.62, 145.16, 139.8, 136.39, 136.22, 130.18, 130.14, 128.23, 128.0, 126.97, 122.91, 113.28, 72.1, 71.04, 56.7, 56.3, 56.19, 48.2, 46.07, 42.96, 42.32, 40.65, 40.41, 36.70, 36.07, 35.87, 35.57, 34.79, 32.13, 31.92, 30.77, 30.37, 29.83, 29.8, 29.78, 29.72, 29.68, 29.4, 28.47, 28.3, 28.03, 27.41, 27.29, 27.13, 27.09, 26.63, 24.45, 23.59, 22.9, 21.05, 19.41, 18.76, 18.48, 14.32, 12.28.
Synthesis of Extended Steroid Conjugates Immobilized on Solid Support with Hydroxyl-Prolinol Linker (87)
Succinate 86 (1 g, 0.649 mmol) was dissolved in dichloroethane (3 mL). To that solution DMAP (0.079 g, 0.649 mmol) was added. 2,2′-Dithio-bis(5-nitropyridine) (0.202 g, 0.649 mmol) in acetonitrile/dichloroethane (3:1, 3 mL) was added successively. To the resulting solution triphenylphosphine (0.17 g, 0.65 mmol) in acetonitrile (1.5 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) was added. The suspension was agitated for 16 h. The CPG was filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The loading capacity of the CPG was measured by taking UV measurement. (62 μM/g).
Synthesis of Extended Steroid Conjugates with Hydroxyl-Prolinol Linker
Synthesis of 4-(3-Hydroxy-10,13-dimethyl-hexadecahydro-cyclopenta[a]phenanthren-17-yl)-pentanoic acid octadecylamide (88)
Lithocholic acid (81) (9.78 g, 26 mmol) was dissolved in anhydrous tetrahydrofuran (60 mL). Isobutylchloroformate (3.55 g, 3.55 mL, 26 mmol) was added followed by the addition of triethylamine (5.26 g, 7.25 mL, 52 mmol) and dioctadecylamine (7 g, 26 g). The reaction mixture was brought to ambient temperature and allowed to stir over night. The reaction mixture was concentrated under vacuum, and the residue was dissolved in dichloromethane (250 mL).
The organic layer was washed with 5% sodium bicarbonate, 3% aqueous HCl and water. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure to afford amide 88 (14.5 g) in 89% yield. This was used as such for the next step.
Synthesis of Carbonic acid 10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-yl ester 2,5-dioxo-pyrrolidin-1-yl ester (89)
Referring to scheme 22, amide 88 (14.5 g, 23 mmol) was dissolved in anhydrous dichloromethane (150 mL). To the solution were added disuccinimidyl carbonate (8.87 g, 34 mmol), triethylamine (15 mL) and acetonitrile (50 mL). The reaction mixture was stirred at room temperature under argon for 6h and then evaporated dryness. The residue was dissolved in dichloromethane (300 mL). It was washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer was dried over Na2SO4, filtered and evaporated to dryness. Compound 89 (14.3 g, 81%) was obtained as colorless powder after drying in high vacuum, which was directly used for the next step without further purification.
Synthesis of (6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-yl ester 90)
Amine 5 (4.22 g, 7.9 mmol) is dissolved in anhydrous dichloromethane (25 mL) and cooled to 0° C. To the solution are added pyridine (10 mL) and compound 89 (9.3 g, 7.9 mmol) successively. The reaction temperature was brought to ambient temperature and stirred further. The completion of the reaction is ascertained by TLC (EtOAc). The reaction mixture is diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer is dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 90 is obtained as a white solid after column chromatography over silica gel.
Synthesis of extended steroid conjugates phosphoramidite with hydroxyl-prolinol linker (91)
Compound 90 (4.75 g, 4 mmol) is coevaporated with anhydrous toluene (50 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.225 g, 2 mmol) is added and the mixture is dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.8 g, 1.97 mL, 6 mmol) is added. The reaction mixture is stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted with dichloromethane (100 mL) and washed with 5% NaHCO3 (100 mL) and brine (100 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 91.
Synthesis of (6-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 10,13-dimethyl-17-(1-methyl-3-octadecylcarbamoyl-propyl)-hexadecahydro-cyclopenta[a]phenanthren-3-yl ester (2)
Referring to scheme 23, Compound 90 (1.185 g, 1 mmol) is mixed with succinic anhydride (0.15 g, 1.5 mmol) and DMAP (0.0122 g, 0.1 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.101 g, 0.14 mL, 1 mmol) is added and the solution is allowed to stir at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (50 mL) and washed with ice cold aqueous citric acid (5% wt., 25 mL) and water (2×25 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The crude product was purified by column chromatography to afford compound 92.
Synthesis of Extended Steroid Conjugates Immobilized on Solid Support with Hydroxyl-Prolinol Linker (93)
Succinate 92 (0.833 g, 0.649 mmol) is dissolved in dichloroethane (3 mL). To that solution DMAP (0.079 g, 0.649 mmol) is added. 2,2′-Dithio-bis(5-nitropyridine) (0.202 g, 0.649 mmol) in acetonitrile/dichloroethane (3:1, 3 mL) is added successively. To the resulting solution triphenylphosphine (0.17 g, 0.65 mmol) in acetonitrile (1.5 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (2.5 g, 155 μm/g) is added. The suspension is agitated further. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The loading capacity of the CPG is measured by taking UV measurement.
Synthesis of Dimethylamino Phosphoramidite with Hydroxyl-Prolinol Linker
1-{2-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-dimethylamino-hexan-1-one (94)
Hydrochloride salt of 5-dimethylamino-pentanoic acid (1.95 g, 10 mmol) is suspended in anhydrous pyridine (50 mL). To the suspension is added diisopropylcarbodiimide (1.262 g, 1.55 mL, 10 mmol) followed by amine 38 (4.2 g, 10 mmol). The stirring is allowed to continue for 16 h. The reaction mixture is concentrated under vacuum, to the residue ethyl acetate is added and washed with, 5% NaHCO3 solution, brine and water. After drying over anhydrous sodium sulfate the solvent is removed to afford crude product. DMT-alcohol 94 is obtained after purification over silica gel.
Synthesis of Dimethylamino Phosphoramidite with Hydroxyl-Prolinol Linker (95)
Compound 94 (2.35 g, 4.2 mmol) is coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g, 2.1 mmol) is added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) is added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 95.
Synthesis of Succinic Acid mono-[5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-(6-dimethylamino-hexanoyl)-pyrrolidin-3-yl] Ester (96)
Referring to scheme 25, Compound 94 (1.2 g, 2 mmol) is mixed with succinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.476 g, 0.64 mL, 4 mmol) is added and the solution stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The crude product is purified by column chromatography to afford compound 96.
Synthesis of Dimethylamino Immobilized Solid Support with Hydroxyl-Prolinol Linker (97)
Succinate 96 (1 g, 1.5 mmol) is dissolved in dichloroethane (7 mL). To that solution DMAP (0.183 g, 1.5 mmol) is added. 2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1.5 mmol) in acetonitrile/dichloroethane (3:1, 7 mL) is added successively. To the resulting solution triphenylphosphine (0.395 g, 1.5 mmol) in acetonitrile (3 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using wrist-action shaker (5 mins).
Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added. The suspension was agitated for 4 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The loading capacity of the CPG is measured by taking UV measurement.
Synthesis of Nalidixic Phosphoramidite with Hydroxyl-Prolinol Linker
1-Ethyl-7-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carboxylic Acid (6-{2-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-amide (99)
Nalidixic acid (2.32 g, 10 mmol) is suspended in anhydrous pyridine (50 mL). To the suspension is added diisopropylcarbodiimide (1.262 g, 1.55 mL, 10 mmol) followed by amine 5 (5.32 g, 10 mmol). The stirring is allowed to continue for 16 h. The reaction mixture is concentrated under vacuum, to the residue ethyl acetate is added and washed with, 5% NaHCO3 solution, brine and water. After drying over anhydrous sodium sulfate the solvent is removed to afford crude product. DMT-alcohol 99 is obtained after purification over silica gel.
Synthesis of Nalidixic Phosphoramidite with Hydroxyl-Prolinol Linker (100)
Compound 99 (3.12 g, 4.2 mmol) is coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g, 2.1 mmol) is added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) is added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 100.
Synthesis of Succinic Acid mono-(5-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[(1-ethyl-7-methyl-4-oxo-1,4-dihydro-[1,8]naphthyridine-3-carbonyl)-amino]-hexanoyl}-pyrrolidin-3-yl) Ester (101)
Referring to scheme 27, Compound 99 (1.48 g, 2 mmol) is mixed with succinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.606 g, 0.96 mL, 6 mmol) is added and the solution stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The crude product is purified by column chromatography to afford compound 101.
Synthesis of Nalidixic Immobilized Solid Support with Hydroxyl-Prolinol Linker 102)
Succinate 100 (1.26 g, 1.5 mmol) is dissolved in dichloroethane (7 mL). To that solution DMAP (0.183 g, 1.5 mmol) is added. 2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1.5 mmol) in acetonitrile/dichloroethane (3:1, 7 mL) is added successively. To the resulting solution triphenylphosphine (0.395 g, 1.5 mmol) in acetonitrile (3 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added. The suspension was agitated for 4 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The loading capacity of the CPG is measured by taking UV measurement.
Diosgenein Phosphoramidite with Hydroxyl-Prolinol Linker
Diosgenin Succinimidyl Carbamate (104)
Referring to scheme 28, diosgenin (6.9 g, 16.74 mmol) is dissolved in anhydrous dichloromethane (150 mL). To the solution are added disuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine (10 mL) and acetonitrile (50 mL). The reaction mixture is stirred at room temperature under argon for 6h and then evaporated dryness. The residue is dissolved in dichloromethane (300 mL). It is washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer is dried over Na2SO4, filtered and evaporated to dryness. Compound 104 is obtained as colorless powder after drying in high vacuum, which is directly used for the next step without further purification.
Synthesis of Diosgenin DMT-Alcohol 105
Amine 5 (10.5 g, 19.7 mmol) is dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL) and compound 104 (9.62 g, 17.3 mmol) successively. The reaction temperature is brought to ambient temperature and stirred further for 3h. The completion of the reaction is ascertained by TLC (10% MeOH/CHCl3). The reaction mixture is diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer is dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 105 is obtained as a white solid after column chromatography over silica gel.
Synthesis of Diosgenin Phosphoramidite with Hydroxyl-Prolinol Linker (106)
Compound 105 (4.1 g, 4.2 mmol) is coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g, 2.1 mmol) is added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) is added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 106.
Synthesis of Diosgenin-Hydroxy-Prolinol Succinate 107
Referring to scheme 29, Compound 105 (1.95 g, 2 mmol) is mixed with succinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.676 g, 0.96 mL, 6 mmol) is added and the solution stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The crude product is purified by column chromatography to afford compound 107.
Synthesis of Diosgenin Immobilized Solid Support with Hydroxyl-Prolinol Linker 108)
Succinate 107 (1.61 g, 1.5 mmol) is dissolved in dichloroethane (7 mL). To that solution DMAP (0.183 g, 1.5 mmol) is added. 2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1.5 mmol) in acetonitrile/dichloroethane (3:1, 7 mL) is added successively. To the resulting solution triphenylphosphine (0.395 g, 1.5 mmol) in acetonitrile (3 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added. The suspension was agitated for 4 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The loading capacity of the CPG is measured by taking UV measurement.
Epifriedelanol Phosphoramidite with Hydroxyl-Prolinol Linker
Epifriedelanol Succinimidyl Carbamate (110)
Referring to scheme 30, Epifriedelanol (7.2 g, 16.74 mmol) is dissolved in anhydrous dichloromethane (150 mL). To the solution are added disuccinimidyl carbonate (6.4 g, 25.1 mmol), triethylamine (10 mL) and acetonitrile (50 mL). The reaction mixture is stirred at room temperature under argon for 6h and then evaporated dryness. The residue is dissolved in dichloromethane (300 mL). It is washed with saturated NaHCO3 aqueous solution (3×100 mL). The organic layer is dried over Na2SO4, filtered and evaporated to dryness. Compound 110 is obtained as colorless powder after drying in high vacuum, which is directly used for the next step without further purification.
Synthesis of Epifriedelanol DMT-Alcohol 111
Amine 5 (10.5 g, 19.7 mmol) is dissolved in anhydrous dichloromethane (50 mL) and cooled to 0° C. To the solution were added pyridine (10 mL) and compound 110 (9.85 g, 17.3 mmol) successively. The reaction temperature is brought to ambient temperature and stirred further for 3h. The completion of the reaction is ascertained by TLC (10% MeOH/CHCl3). The reaction mixture is diluted with dichloromethane and washed with saturated NaHCO3, water followed by brine. The organic layer is dried over sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Compound 111 is obtained as a white solid after column chromatography over silica gel.
Synthesis of Epifriedelanol Phosphoramidite with Hydroxyl-Prolinol Linker (112)
Compound 111 (4.14 g, 4.2 mmol) is coevaporated with anhydrous toluene (25 mL). To the residue N,N-tetraisopropylammonium tetrazolide (0.238 g, 2.1 mmol) is added and the mixture was dried over P2O5 in a vacuum oven for overnight at 40° C. The reaction mixture is dissolved in dichloromethane (25 mL) and 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.9 g, 2.1 mL, 6.3 mmol) is added. The reaction mixture was stirred at ambient temperature for overnight. The completion of the reaction is ascertained by TLC (1:1 ethyl acetate:hexane). The reaction mixture is diluted with dichloromethane (50 mL) and washed with 5% NaHCO3 (50 mL) and brine (50 mL). The organic layer is dried over anhydrous Na2SO4 filtered and concentrated under reduced pressure. The residue was purified over silica gel (50:49:1, EtOAc:Hexane:triethlyamine) to afford 112.
Synthesis of Epifriedelanol-Hydroxy-Prolinol Succinate 113
Referring to scheme 31, Compound 111 (1.975 g, 2 mmol) is mixed with succinic anhydride (0.200 g, 2 mmol) and DMAP (0.244 g, 13 mmol) and dried in a vacuum at 40° C. overnight. The mixture is dissolved in anhydrous dichloromethane (5 mL), triethylamine (0.676 g, 0.96 mL, 6 mmol) is added and the solution stirred at room temperature under argon atmosphere for 16 h. It is then diluted with dichloromethane (100 mL) and washed with ice cold aqueous citric acid (5% wt., 100 mL) and water (2×100 mL). The organic phase is dried over anhydrous sodium sulfate and concentrated to dryness. The crude product is purified by column chromatography to afford compound 113.
Synthesis of Epifriedelanol Immobilized Solid Support with Hydroxyl-Prolinol Linker (114)
Succinate 113 (1.63 g, 1.5 mmol) is dissolved in dichloroethane (7 mL). To that solution DMAP (0.183 g, 1.5 mmol) is added. 2,2′-Dithio-bis(5-nitropyridine) (0.470 g, 1.5 mmol) in acetonitrile/dichloroethane (3:1, 7 mL) is added successively. To the resulting solution triphenylphosphine (0.395 g, 1.5 mmol) in acetonitrile (3 ml) is added. The reaction mixture turned bright orange in color. The solution is agitated briefly using wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (4 g, 155 μm/g) is added. The suspension was agitated for 4 h. The CPG is filtered through a sintered funnel and washed with acetonitrile, dichloromethane and ether successively. Unreacted amino groups are masked using acetic anhydride/pyridine. The loading capacity of the CPG is measured by taking UV measurement.
Compound 203a:
The ester 203a was prepared according to reported procedure from the literature (Org. Syn., 1984, 63, 183). Naproxen (201a 10.00 g, 43.427 mmol, purchased from Aldrich) and 4-(Dimethylamino)pyridine (DMAP, 0.53 g, 4.338 mmol, purchased from Aldrich) were dissolved in anhydrous N,N-dimethylformamide (DMF) and 1,3-diisopropylcarbodiimide (DICC, 6.8 mL, 43.914 mmol, purchased from Aldrich) was added into the solution and stirred at ambient temperature for 5 minute. 6-aminohexanoic acid methyl ester hydrochloride (202, 10.00 g, 57.408 mmol, purchased from Fluka) and diisopropylethylamine (DIEA, 10 mL, purchased from Aldrich) were added into the stirring solution after 5 minute of addition of DICC and stirred overnight at ambient temperature. DMF was removed from the reaction in vacuo, the product was extracted into ethyl acetate (EtOAc, 200 mL), washed successively with aqueous KHSO4, water, sodium bicarbonate solution and water. The organic layer was dried over anhydrous sodium sulfate (Na2SO4) and filtered. A white solid was precipitated out from the EtOAc extract by adding hexane to afford the desired compound 203a, 11.20 g (72.14%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.95-7.92 (t, J(H,H)=5.2 & 5.6 Hz, 1H), 7.76-7.68 (m, 3H), 7.43-7.40 (dd, J′(H,H)=1.6 and J″(H,H)=8.4 Hz, 1H), 7.25-7.24 (d, J(H,H)=2.0 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.4 and J″(H,H)=8.8 Hz, 1H), 3.84 (s, 3H), 3.70-3.65 (q, J(H,H)=6.8 and 7.2 Hz, 1H), 3.54 (s, 3H), 3.00-2.97 (q, J(H,H)=6.8 Hz, 2H), 2.21-2.17 (t, 2H), 1.48-1.29 (m, 7H), 1.19-1.13 (m, 2H).
Compound 204a:
Hydrolysis of the ester 203a was performed as reported earlier (Rajeev et al., 2002, 4, 4395). Compound 203a (10.80 g, 30.24 mmol) was suspended in tetrahydrofuran-water (THF-H2O) mixture (4:1, 40 mL) and stirred with LiOH (1.65 g, 39.32 mmol) for 4 h at ambient temperature. THF was removed from the reaction in vacuo and free acid was precipitated out from water by adding concentrated KHSO4 solution, thoroughly washed with water, filtered through a sintered filter, triturated with diethyl ether and dried over P2O5 under vacuum overnight to obtain the acid 204a as a white solid, 10.22 g (98.4%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 11.96 (bs, 1H), 7.95-7.92 (t, J(H,H)=5.37 Hz, 1H), 7.77-7.68 (m, 3H), 7.43-7.41 (d, J(H,H)=8.3 Hz, 1H), 7.25-7.24 (d, J(H, H)=2.44 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=1.95, 2.44 and J″(H,H)=8.79, 9.27 Hz, 1H), 3.84 (s, 3H), 3.71-3.65 (q, J(H,H)=6.84, 7.33 Hz, 1H), 3.02-2.97 (m, 2H), 2.13-2.09 (t, J(H,H)=7.33 Hz, 2H), 1.46-1.30 (m, 7H), 1.21-1.15 (m, 2H).
Compound 205a:
Compound 204a (5.00 g, 14.57 mmol), DMAP (0.18 g, 1.47 mmol) and pentafluorophenol (3.50 g, 19.02 mmol, purchased from Aldrich) were taken in dichloromethane (40 mL) and DCC (3.00 g, 14.54 mmol) was added into the solution. Reaction mixture was stirred at ambient temperature for 8 h. The reaction mixture was diluted to 100 mL by adding EtOAc and precipitated DCU was removed by filtration. Combined filtrate, evaporated solvent in vacuo, and the residue was subsequently filtered through a column of silica gel, eluent hexane/EtOAc 4:1 to obtain a mixture (7.90 g) of the desired ester 205a and excess pentafluorophenol from the reaction. The crude product thus obtained was directly used for proceeding experiments without further purification.
Compound 206a:
Pentafluorophenol ester 205a was stirred with serinol in the presence of TEA to obtain compound 206a (J. Org. Chem., 1991, 56, 1713). Compound 205a (4.00 g, 7.86 mmol) and serinol (1.5 g, 16.46 mmol, purchased from Aldrich) were suspended in dichloromethane (30 mL) and triethylamine (TEA, 2.3 mL, purchased from Aldrich) was added into the suspension, stirred at ambient temperature for 2 h. A white precipitate was formed during the course of the reaction. After 2 h, the precipitate was filtered through a sintered filter, washed successively with excess of dichloromethane, water and diethyl ether to afford desired product 206a (2.82 g, 86.2%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.95-7.92 (t, J(H, H)=5.49 Hz, 1H, exchangeable with D2O), 7.77-7.68 (m, 3H), 7.43-7.39 (m, 2H, accounted for 1H after D2O exchange), 7.26-7.25 (d, J(H,H)=2.14 Hz, 1H), 7.13-7.11 (dd, J′(H,H)=2.44 and J″(H,H)=8.85 Hz, 1H), 4.58-4.55 (t, J(H,H)=5. 49 Hz, 2H, exchangeable with D2O), 3.84 (s, 3H), 3.71-3.65 (m, 2H), 3.37-3.35 (t, became doublet after D2O exchange, 4H), 3.02-2.95 (m, 2H), 2.03-2.01 (t, J(H,H)=7.32, 7.63 Hz, 2H), 1.46-1.30 (m, 7H), 1.20-1.12 (m, 2H).
Compound 207a:
Compound 206a was prepared by modifying reported literature procedure (Rajeev et al., Org. Lett., 2003, 5, 3005). A solid mixture of compound 206a (2.50 g, 6.01 mmol) and DMAP (0.075 g, 0.61 mmol) was dried over P2O5 under vacuum overnight. The solid mixture was suspended in anhydrous pyridine (100 mL) under argon and heated to obtain a homogenous solution. The temperature of the mixture was brought to room temperature and stirred. 4,4′-Di-O-methyltrityl chloride (2.24 g, 6.61 mmol, purchased from Chem Genes Corporation) was separately dissolved in 20 mL of anhydrous dichloromethane and added drop-wise into the stirring pyridine solution over a period of 45 minute under argon. Reaction mixture was further stirred overnight. Solvents were removed form the reaction mixture and the product was extracted into EtOAc (150 mL) and washed successively with water, NaHCO3 solution and water, dried over anhydrous Na2SO4 and evaporated to solid mass. Desired product was purified by flash silica gel column chromatography: (a) eluent: 1% methylalcohol (MeOH) in dichloromethane—1.60 g of undesired bis DMT derivative (26. 1%) and (b) 5% MeOH in dichloromethane—2.50 g of desired product 207a (57.9%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.94-7.91 (t, J(H,H)=5.49 Hz, 1H, exchangeable with D2O), 7.7-7.68 (m, 3H), 7.60-7.58 (d, J(H,H)=8.55 Hz, 1H, exchangeable with D2O), 7.43-7.10 (m, 12H), 6.86-6.84 (d, 4H), 4.62-4.59 (t, J(H,H)=5.18, 5.49 Hz, 1H, exchangeable with D2O), 4.01-3.96 (m, 1H), 3.83 (s, 3H), 3.71-3.65 (m, 7H), 3.44-3.42 (t, J(H,H)=5.19, 5.49 Hz, 2H), 3.03-2.87 (m, 4H), 2.05-2.01 (t, J(H,H)=7.33, 7.63 Hz, 2H), 1.48-1.30 (m, 7H), 1.21-1.14 (m, 2H).
Compound 208a:
The desired solid support 208a was prepared according to reported procedures (References for succinilation: Rajeev et al., Org. Lett., 2003, 5, 3005 and for conjugation to CPG: Kumar et al., Nucleosides Nucleotides, 1996, 15, 879). A mixture of compound 207a (1.00 g, 1.39 mmol), succinic anhydride (0.17 g, 1.69 mmol, purchased from Aldrich) and DMAP (0.21 g, 1.72 mmol) were suspended in 7 mL of anhydrous ethylene dichloride for 24 h. Reaction mixture was diluted to 50 mL by adding dichloromethane and washed with dilute aqueous citric acid solution (20 mL), dried over anhydrous Na2SO4 and evaporated to dryness. The residue obtained was further dried over P2O5 under vacuum to afford an almost pure but crude monosuccinate as a white solid (1.10 g, 96.5%). The product obtained was directly used for subsequent reaction without further purification. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.94-7.91 (t, J(H,H)=5.19, 5.49 Hz, 1H, exchangeable with D2O), 7.83-7.81 (d, J(H,H)=7.94 Hz, 1H, exchangeable with D2O), 7.76-7.68 (m, 3H), 7.42-7.10 (m, 12H), 6.88-6.86 (d, 4H), 4.18-4.12 (m, 2H), 4.07-3.98 (m, 2H), 3.83 (s, 3H), 3.71-3.66 (m, 7H), 3.00-2.91 (m, 4H), 2.40 (s, 4H), 2.04-2.00 (t, J(H,H)=7.32 Hz, 2H), 1.44-1.22 (m, 7H), 1.19-1.15 (m, 2H).
2,2′-Dithiobis(5-nitropyridine) (0.38 g, 1.22 mmol, DTNP, purchased from Adrich) was dissolved in a 1:1 mixture of acetonitrile and ethylene dichloride (5 mL) and added into a suspension of naproxen—6-aminohexanoic acid—serinol conjugate mono DMT mono succinate (1.00 g, 1.21 mmol) and DMAP (0.16 g, 1.31 mmol) in 2 mL of anhydrous acetonitrile. Triphenylphosphine (Ph3P, 0.32 g, 1.22 mmol, purchased from Aldrich) was added into the reaction mixture and shaken for 3-4 minute. 5.5 g of long chain aminoalkyl controlled pore glass (CPG) with 500A size and a loading of 112.7 μM/g (purchased from Millipore), and excess of acetonitrile (to soak the CPG completely) were added into the reaction mixture and the suspension was shaken (agitated) for 45 minute at ambient temperature. CPG was filtered through a sintered funnel, washed extensively with acetonitrile, dichloromethane and diethyl ether and subsequently re-suspended in pyridine-dichloromethane and treated with acetic anhydride in the presence of DIEA to cap unreacted amino groups on the CPG. After 10 minute, CPG was filtered and extensively washed with dichloromethane, acetonitrile and diethyl ether followed by drying under vacuum to obtain the desired CPG 208a with a loading 54.12 μM/g. The loading was determined as reported in the literature (Prakash et al., J. Org. Chem., 2002, 67, 357 and references cited therein).
Compound 209a:
The phosphoramidite was prepared as reported in the literature (Rajeev et al., Org. Lett., 2003, 5, 3005 and references cited therein). Compound 207a (1.00 g, 1.39 mmol) and diisopropylammonium tetrazolide (0.12 g, 0.70 mmol) were dried over P2O5 vacuum overnight and subsequently suspended in anhydrous acetonitrile (5 mL) under argon atmosphere. 2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.69 mL, 2.09 mmol) was added into the suspension and stirred at ambient temperature for 14 h. Solvent was removed form the reaction in vacuo and residue was suspended in EtOAc (40 mL) and washed with dilute NaHCO3 solution followed by standard work. Desired amidite 209a was purified by flash silica gel column chromatography; eluent: 100 EtOAc, yield 0.79 g (61.8%). 31P NMR (161.8 MHz, CDCl3, 25° C.): δ 146.01, 145.69.
Compound 201c:
Naproxen (201, 11.25 g, 48.86 mmol), pentafluorophenol (10.00 g, 54.33 mmol) and DMAP (0.60 g, 4.91 mmol) were dissolved in DMF (40 mL) and stirred at ambient temperature. 1,3-dicyclohexylcarbodiimide (DCC, 11.00 g, 53.31 mmol) was added into the solution and continued stirring overnight. 1,3-dicyclohexylurea (DCU) was precipitated out during the course of the reaction. The precipitated DCU was filtered off, washed with DMF, combined filtrate and removed DMF in vacuo. Oily residue obtained was filtered through a small column of silica gel, eluent 10% EtOAc in hexane to remove dissolved DCU to afford a mixture of the desired ester 201c and excess pentafluorophenol (20.30 g). The crude product thus obtained was directly used for proceeding experiments without further purification. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.85-7.81 (m, 3H), 7.48-7.46 (dd, J′(H,H)=1.53 and J″(H,H)=8.55 Hz, 1H), 7.32-7.31 (d, J(H,H)=2.44 Hz, 1H), 7.18-7.16 (dd, J′(H,H)=2.44 and J″(H,H)=8.85 Hz, 1H), 4.47-4.44 (q, J(H,H)=7.02 Hz), 3.86 (s, 3H), 1.63-1.61 (d, J(H,H)=7.34 Hz, 3H).
Compound 203b:
Ibuprofen (201b, 5.0 g, 24.23 mmol, purchased from Acros Organic), methyl 6-aminohexanoic acid monohydrochloride (202, 6.60 g, 36.33 mmol, purchased from Fluka) and DMAP (0.30 g, 2.46 mmol) were suspended in dichloromethane (60 mL) in a 200 mL round bottom flask and DCC (5.00 g, 24.23 mmol) was added into the suspension, stirred for 3 minute. After 3 minute, 3.6 mL (25.83 mmol) of TEA was added into the reaction and continued stirring at ambient temperature for 18 h. Solvent and excess TEA were removed from the reaction in vacuo and residue obtained was triturated with diethyl ether, filtered through a sintered funnel to remove DCU. Combined filtrate and evaporated on a rotary evaporator. Residue was redissolved in EtOAc (100 mL) and successively washed with KHSO4 solution, water, NaHCO3 solution and water followed by drying over anhydrous Na2SO4 and evaporation of solvent in vacuo to obtain yellowish viscous residue of compound 203b (8.0 g). The crude product thus obtained was directly used for subsequent reaction without further purification. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.86-7.84 (bt, J(H,H)=5.39, 5.00 Hz, 1H, exchangeable with D2O), 7.19-7.03 (m, 4H), 3.56 (s, 3H), 3.53-3.47 (q, J(H,H)=7.05 Hz, 1H), 3.00-2.95 (q, J(H,H)=6.64, 5.81 Hz, 2H), 2.39-2.37 (m, 2H, mixture of rotamers), 2.23-2.20 (t, J(H,H)=7.45, 7.05 Hz, 2H), 1.81-1.74 (m, 1H), 1.49-1.41 (m, 2H), 1.36-1.26 (m, 5H), 1.19-1.11 (m, 2H), 0.84-0.82 (m, 6H, mixture of rotamers).
Compound 204b:
Compound 203b (8.00 g, 24.01 mmol) was stirred with LiOH (1.21 g, 28.84 mmol) in THF-H2O (4:1, 40 mL) for 4 h. Solvents were removed from the reaction mixture in vacuo and the residue was washed with concentrated KHSO4 solution. Unlike the corresponding naproxen analogue 204a, the free acid 204b did not precipitate out from the aqueous phase, so the aqueous phase was repeatedly extracted with EtOAc, combined extract, dried over Na2SO4 and evaporated in vacuo to obtain slightly yellowish viscous residue, 6.60 g (86.1%). The acid 204b thus obtained was directly used for subsequent experiments without further purification. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 11.96 (bs, 1H, exchangeable with D2O), 7.87-7.84 (t, J(H,H)=5.39 Hz, 1H, exchangeable with D2O), 7.19-7.04 (m, 4H), 4.04-3.99 (q, J(H,H)=7.05 Hz, 1H), 3.62-3.57 (q, J(H,H)=7.05 Hz, 0.1H, minor rotamer), 3.53-3.47 (q, J(H,H)=7.05 Hz, 1.9H), 3.00-2.95 (q, J(H,H)=6.22 Hz, 2H), 2.41-2.37 (m, 2H, mixture of rotamers), 2.14-2.10 (t, J(H,H)=7.47, 7.05 Hz, 2H), 1.81-1.74 (m, 1H), 1.46-1.40 (m, 2H), 1.36-1.26 (m, 5H), 1.20-1.12 (m, 2H), 0.85-0.82 (m, 6H, mixture of rotamers).
Compound 206b:
Compound 204b (6.60 g, 20.676 mmol), DMAP (0.26 g, 2.128 mmol) and pentafluorophenol (5.70 g, 30.97 mmol) were dissolved in dichloromethane (60 mL) and DCC (4.27 g, 20.70 mmol) was added into the stirring solution. The reaction mixture was allowed to stir for 8 h. Precipitated DCU was removed by filtration and the filtrate was evaporated to obtain a crude oil containing the desired ester 205b. The crude 205b thus obtained was stirred with serinol (3.5 g, 38.42 mmol) in dichloromethane in the presence of TEA (8 mL) for 2 h. A white precipitate was formed during the course of the reaction, which was filtered washed successively with dichloromethane, water and diethyl ether and dried over P2O5 to obtain 2.4 g of the product 206b. Extraction of the aqueous phase with EtOAc afforded another 1.05 g of the desired product 206b. Combined yield was 42.5%. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.87-7.84 (t, J(H,H)=5.86, 5.37 Hz, 1H, exchangeable with D2O), 7.42-7.40 (d, J(H,H)=7.81 Hz, 1H, exchangeable with D2O), 7.19-7.17 (d, J(H,H)=8.30 Hz, 2H), 7.06-7.04 (d, J(H,H)=8.30 Hz, 2H), 4.57 (bs, 2H, exchangeable with D2O), 3.69-3.63 (m, 1H), 3.53-3.47 (q, J(H,H)=6.83 Hz, 1H), 3.36-3.34 (d, J(H,H)=5.37 Hz, 4H), 3.02-2.91 (m, 2H), 2.39-2.37 (d, J(H,H)=7.34 Hz, 2H), 2.04-2.00 (t, J(H,H)=7.33 Hz, 2H), 1.81-1.75 (m, 1H), 1.44-1.26 (m, 7H), 1.18-1.12 (m, 2H), 0.84-0.83 (d, J(H,H)=6.35 Hz, 6H).
Compound 207b:
A solid mixture of compound 206b (3.00 g, 7.65 mmol), 4,4′-dimethoxytrityl chloride (2.85 g, 8.41 mmol) and DMAP (0.20 g, 1.64 mmol) was taken in a 200 mL RB and dried over P2O5 under vacuum overnight. Anhydrous pyridine (40 mL) was added into the mixture under argon and stirred for overnight. Pyridine was removed from the reaction and residue was suspended in EtOAc (100 mL) followed by standard workup. Desired mono DMT and bis DMT products were separated by flash silica gel column chromatography, eluent: 2-3% methanol in dichloromethane, 170 g (22.3%, bis DMT derivative) and eluent: 4% methanol in dichloromethane, 1.89 g (35.6%, desired mono DMT product 207b). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.83-7.80 (t, J(H,H)=5.37 Hz, 1H, exchangeable with D2O), 7.58-7.55 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D2O), 7.34-7.32 (d, J(H,H)=7.33 Hz, 2H), 7.26-7.14 (m, 9H), 7.02-7.00 (d, J(H,H)=7.81 Hz, 2H), 6.83-6.81 (d, J(H,H)=8.79 Hz, 4H), 4.58-4.56 (t, J(H,H)=5.37, 4.88 Hz, 1H, exchangeable with D2O), 3.95-3.93 (m, 1H), 3.68 (s, 6H), 3.48-3.45 (q, J(H,H)=7.34 Hz, 1H), 3.41-3.38 (t, J(H,H)=5.37 Hz, 2H), 2.96-2.84 (m, 4H), 2.34-2.33 (d, J(H,H)=7.33 Hz, 2H), 2.02-1.98 (t, J(H,H)=7.33, 7.81 Hz, 2H), 1.76-1.69 (m, 1H), 1.44-1.36 (m, 2H), 1.33-1.23 (m, 5H), 1.16-1.08 (m, 2H), 0.80-0.78 (d, J(H,H)=6.35 Hz, 6H). 13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 174.0, 172.8, 158.3, 145.4, 139.9, 139.7, 136.2, 130.1, 129.2, 128.2, 128.1, 127.3, 113.5, 85.5, 61.0, 55.4, 51.1, 45.1, 44.6, 35.7, 30.0, 29.1, 26.3, 25.4, 22.5, 18.8.
Compound 208b:
The desired succinate (0.98 g, 85.7%) was synthesized from the corresponding precursor 207b (1.00 g, 1.44 mmol), DMAP (0.27 g, 2.21 mmol) and succinic anhydride (0.22 g, 2.20 mmol) as described for the corresponding naproxen derivative. The succinic acid derivative was purified by flash silica gel column chromatography, eluent: 5% methanol in dichloromethane. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.86-7-80 (m, 2H, exchangeable with D2O), 7.34-7.32 (d, J(H,H)=7.33 Hz, 2H), 7.28-7.13 (m, 9H), 7.02-7.00 (d, J(H,H)=8.30 Hz, 2H), 6.85-6.83 (d, J(H,H)=8.79 Hz, 4H), 4.14-1.10 (bm, 2H), 4.02-3.98 (m, 1H), 3.68 (s, 6H), 3.50-3.44 (q, J(H,H)=7.33, 6.83 Hz, 2H), 2.96-2.87 (m, 2H), 2.35-2.33 (m, 6H), 2.51-2.45 (m, 7H, 2H+DMSO-d6), 2.01-1.96 (t, J(H,H)=7.32 Hz, 2H), 1.77-1.69 (m, 1H), 1.42-1.22 (m, 7H), 1.15-1.07 (m, 2H), 0.80-0.78 (d, J(H,H)=6.35 Hz, 6H). 13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 174.9, 174.3, 173.2, 158.5, 145.3, 139.9, 139.8, 136.0, 130.2, 129.3, 128.4, 128.1, 127.4, 113.6, 85.8, 55.5, 46.1, 46.1, 45.3, 44.7, 35.6, 30.1, 29.0, 26.2, 25.4, 22.6, 18.8.
The desired CPG 208b (4.50 g) with a loading capacity of 85.62 μM/g was prepared from 0.92 g (1.16 mmol) of the ibuprofen succinate thus obtained, 2,2′-Dithiobis(5-nitropyridine) (0.37 g, 1.18 mmol), DMAP (0.15 g, 1.23 mmol), Ph3P (0.31 g, 1.18 mmol) and long chain aminoalkyl controlled pore glass (CPG) with 500A size and a loading of 162.5 μM/g as described for the preparation of the corresponding naproxen analogue 208a.
Compound 209b:
The desired amidite 209b is prepared as described for compound 209a in Example 1.
Compound 201d:
Ibuprofen pentafluorophenol ester (201d) was prepared from ibuprofen (201b, 5.00 g, 24.23 mmol), pentafluorophenol (5.4 g, 29.02 mmol), DCC (5.00 g, 24.23 mmol) and DMAP (0.30 g, 2.46 mmol) as described for the synthesis of pentafluorophenol ester (201c) of naproxen (201a).
Compound 210a:
Compound 4 (4.90 g, 7.35 mmol) was dissolved in ethyl acetate-methanol (4:1.16 mL) and purged with argon. To the solution was added 10% palladium on carbon (2 g, wet, Degussa type E101 NE/W). The flask was purged with hydrogen 2 times and stirred further at room temperature under hydrogen at 1 atm for 2h. The disappearance of the starting material was confirmed by TLC analysis. The reaction mixture was filtered through a bed of Celite and washed with ethyl acetate-methanol (4:1). The combined filtrate was concentrated under reduced pressure to afford free amine. The free amine obtained was stirred with compound 101c (3.1 g, 7.82 mmol) in the presence of TEA in dichloromethane (20 mL) for 1 h. The reaction mixture was diluted to 50 mL and washed with aqueous sodium bicarbonate followed by standard workup. Compound 210a was obtained as a white foamy solid after flash silica gel column chromatography, eluent: 3-4% methanol in dichloromethane, yield: 5.45 g (quant.). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.92-7.88 (m, 1H); 7.76-7.68 (m, 3H); 7.43-7.41 (d, J(H,H)=8.5 Hz, 1H); 7.31-7.08 (m, 11H); 6.87-6.83 (m, 4H); 4.97 (bd, 0.7H, exchangeable with D2O); 4.88 (bd, 0.3H, exchangeable with D2O); 4.39-4.35 (m, 0.7H); 4.29-4.26 (m, 0.3H); 4.14-4.10 (m, 0.7H), 3.83-3.82 (d, J(H,H)=2 Hz, 3H, changed to multiplet after D2O exchange); 3.71-3.65 (m, 7H); 3.54-3.50 (m, 0.7H), 3.43-3.40 (m, 0.3H); 3.28-3.22 (m, 1H); 315-3.10 (m, 1H); 3.01-2.95 (m, 3H); 2.12-1.80 (m, 5H); 1.42-1.04 (m, 8H).
Compound 211a:
The solid support 211a is prepared from compound 210a as described in Example 1 for the preparation of compound 208a.
Compound 212a:
The phosphoramidite 212a is prepared from compound 210a as described in Example 1 for the preparation of compound 209a.
Compound 210b:
Compound 210b is obtained from compound 4 and compound 201d as described in
Example 3 for the preparation of compound 210a.
Compound 211b:
The solid support 211b is prepared from compound 210b as described in Example 1 for the preparation of compound 208a.
Compound 212b:
The phosphoramidite 212b is prepared from compound 210b as described in Example 1 for the preparation of compound 209a.
Compound 214a:
DMT-dT-C5-Amino linker (213, 1.00 g, 1.43 mmol) from Chem Genes was stirred with cholesteryl chloroformate (0.77 g, 1.71 mmol) in dichloromethane (10 mL) in the presence of TEA (1.0 mL) at ambient temperature for 2 h. Completion of the reaction was confirmed by TCL monitoring. The reaction mixture was diluted to 50 mL by adding more dichloromethane and washed successively with NaHCO3 solution and water followed by standard workup. Residue obtained was purified by flash silica gel column chromatography to afford 214a (0.66 g, 37.75%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 11.61 (s, 1H, exchangeable with D2-O), 8.01-7.98 (t, J(H,H)=5.39 Hz, 1H, exchangeable with D2O), 7.92 (s, 1H), 7.37-6.99 (m, 12H), 6.87-6.83 (m, 4H), 6.17-6.14 (t, J(H,H)=6.64 Hz, 1H), 5.30 (s, 1H), 5.28-5.27 (d, J(H,H)=4.56 Hz, 1H, exchangeable with D2O), 4.32-4.20 (m, 2H), 3.87-3.84 (m, 1H), 3.71-3.63 (m, 7H), 3.21-3.03 (m, 4H), 2.95-2.88 (m, 2H), 2.33-2.13 (m, 4H), 1.97-1.73 (m, 5H), 1.54-0.82 (m, 40H), 0.63 (s, 3H).
Compound 215a
Compound 214a (0.55 g, 0.495 mmol) and succinic anhydride (0.075 g, 0.749 mmol) were suspended in anhydrous dichloromethane (5 mL) and stirred at ambient temperature in the presence of DMAP (0.18 g, 1.49 mmol) overnight. After confirming completion of the reaction, the reaction mixture was diluted to 50 mL by adding dichloromethane and washed with dilute citric acid solution; organic layer was dried over anhydrous Na2SO4 and evaporated in vacuo. Residue obtained was purified by flash silica gel column chromatography, eluent 6% methanol in dichloromethane, to afford the corresponding succinic acid derivative (0.50 g, 83.4%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 12.24 (bs, 1H, exchangeable with D2O), 11.64 (s, 1H, exchangeable with D2O), 8.02-7.99 (bm, 2H), 7.36-7.00 (m, 12H), 6.87-6.81 (m, 4H), 6.15-6.11 (t, J(H,H)=6.84 Hz, 1H), 5.30 (bs, 1H), 5.17-5.14 (bm, 1H), 4.31-4.24 (m, 1H), 4.05 (bm, 1H), 3.70-3.66 (m, 8H), 3.34-3.08 (m, 6H), 2.94-2.88 (m, 4H), 2.31-2.13 (m, 3H), 1.96-1.71 (bm, 5H), 1.55-0.80 (m, 40H), 0.63 (s, 3H).
The succinnate thus obtained was conjugated to long chain aminoalkyl controlled glass support (CPG) with a loading of 155 μM/g loading as described in the literature by Kumar et al. (Nucleosides and Nucleotides, 1996, 15, 879) to obtain the desired the desired CPG solid support 215a (1.70 g) with a loading of 78.42 μM/g. The loading of the support 215a was determined as described in the literature (Prakash et al., J. Org. Chem., 2002, 67, 357).
Compound 216a
The phosphoramidite 216a is prepared from compound 214a by reacting with 2-Cyanoethyl-N,N, N′, N′-tetraisopropylphosphorodiamidite in the presence of tetrazolediisopropylammonium salt in acetonitrile according to reported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).
Compound 214b
5β-Cholanic acid (5.00 g, 13.87 mmol, purchased from Sigma), pentafluorophenol (2.81 g, 15.27 mmol, purchased from Aldrich) and DMAP (0.20 g, 1.64 mmol) were dissolved in dichloromethane and N,N′-dicyclohexycarbodiimide (DCC, 2.86 g, 13.86 mmol) was added into the solution at ambient temperature. The reaction mixture was stirred for 4h. N,N′-Dicyclohexylurea was filtered off from the reaction and the filtrate was evaporated to obtain pentafluorophenol ester of 5β-cholanic acid. The ester (0.90 g, 1.708 mmol) thus obtained was stirred with compound 213 (1.00 g, 1.431 mmol) in the presence of TEA in dichloromethane (8 mL) for 2 h. The reaction was complete after 2 h as evident from TLC analysis. Reaction mixture was diluted to 50 mL by adding more dichloromethane and washed with dilute NaHCO3 solution followed by standard workup. Residue was purified by flash silica gel column chromatography, eluent 3-4% methanol in dichloromethane, to afford the desired compound 214b (1.46 g, 98.04%).
1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 11.62 (bs, 1H exchangeable with D2O); 8.03-8.00 (t, J(H,H)=5.38 Hz, 1H, exchangeable with D2O), 7.92 (s, 1H), 7.74-7.71 (t, 1H, exchangeable with D2O); 7.37-7.02 (m, 11H), 6.88-6.84 (m, 4H), 6.17-6.14 (t, J(H.H)=6.35, 6.69 Hz, 1H), 4.22-4.19 (m, 1H), 3.88-3.85 (m, 1H), 3.70-3.69 (d, J(H,H)=3.91 Hz, 6H); 3.20-2.89 (m, 6H), 2.33-2.27 (m, 1H), 2.18-2.12 (m, 1H), 2-08-2.00 (m, 1H), 1.99-1.84 (m, 2H), 1.84-1.56 (m, 6H), 1.54-0.94 (m, 33H), 0.87-0.79 (m, 7H), 0.57 (s, 3H).
13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 173.5, 166.1, 162.2, 158.5, 149.7, 145.2, 142.9, 136.0, 135.9, 132.5, 130.1, 128.4, 128.1, 127.2, 122.4, 113.6, 109.8, 107.3, 86.1, 85.9, 85.4, 70.6, 64.2, 56.5, 56.0, 55.4, 55.2, 46.2, 43.5, 42.7, 40.4, 38.7, 37.5, 35.8, 35.3, 32.9, 32.1, 29.5, 29.4, 28.2, 27.5, 27.1, 26.9, 26.6, 26.5, 24.4, 24.3, 21.2, 20.9, 18.6, 12.2, 9.1.
Compound 215b
Compound 215b was prepared from compound 214b as described in Example 1 for the synthesis of compound 215aa. Loading of the support 215b (2.7 g) prepared was determined as 81 μM/g.
Compound 216b
The phosphoramidite 216b is prepared from compound 214b by reacting with 2-Cyanoethyl-N,N, N′, N′-tetraisopropylphosphorodiamidite in the presence of tetrazolediisopropylammonium salt in acetonitrile according to reported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).
Compound 217:
N-Cbz-6-aminohexanoic acid (202, 30.31 g, 114.25 mmol, purchased from Novabiochem), pentafluorophenol (25.00 g, 135.83 mmol) and DMAP (1.54 g, 12.60 mmol) were taken in dichloromethane (100 mL) and to this DCC (26.00 g, 121.01 mmol) added slowly under stirring. During the course of addition, temperature of the reaction rose and dichloromethane started boiling out, so it was cooled down to room temperature and allowed to stir overnight. Reaction mixture was diluted to 200 mL by adding diethyl ether and subsequently filtered through a sintered funnel to remove DCU, washed residue with diethyl ether, combined washing and evaporated to dryness. The desired ester was purified by flash silica gel column chromatography, eluent: hexane/EtOAc 2:1, yield 43.54 g (88.4%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.36-7.23 (m, 6H), 4.99 (s, 2H), 3.01-2.96 (q, J(H,H)=6.35 Hz, 2H), 2.78-2.52 (q, J(H,H)=7.33 Hz, 2H), 1.69-1.61 (m, 2H), 1.47-1.29 (m, 4H).
The pentafluorophenol ester (26.00 g, 60.31 mmol) and serinol (5.00 g, 54.88 mmol) were suspended in 200 mL of dichloromethane and stirred in the presence of TEA (17 mL, 121. 97 mmol) at ambient temperature overnight. A thick white precipitate was formed during the course of the reaction. The reaction mixture was diluted to 200 mL by adding diethyl ether, triturated and filtered. The precipitate was thoroughly washed with diethyl ether and dried under vacuum over P2O5 to obtain 16.51 g (81.0%) of the desired compound 217 as a white solid. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.44-7.42 (d, J(H,H)=7.81 Hz, 1H, exchangeable with D2O), 7.37-7.27 (m, 5H), 7.24-7.20 (t, J(H,H)=5.86, 5.37 Hz, 1H, exchangeable with D2O), 4.99 (s, 2H), 4.58-4.55 (t, J(H,H)=5.37 Hz, 2H, exchangeable with D2O), 3.70-3.65 (m, 1H), 3.37-3.34 (t, J(H,H)=5.86, 3.37 Hz, changed to doublet after D2O exchange, J(H,H, after D2O exchange)=5.37 Hz, 4H), 2.98-2.92 (q, J(H,H)=6.84, 6.35 Hz, 2H), 2.06-2.02 (t, J(H,H)=7.33 Hz, 2H), 1.49-1.33 (m, 4H), 1.24-1.16 (m, 2H).
Compound 218:
Compound 217 (14.10 g, 41.66 mmol) and DMAP 0.60 g, 4.91 mmol) were taken in a 200 mL RB and dried under vacuum over P2O5. The solid mixture then suspended in 50 mL of anhydrous pyridine under argon. 4,4-Dimethoxytrityl chloride (15.5 g, 44.27 mmol) was separately dissolved in 40 mL of anhydrous dichloromethane and added into the stirring pyridine solution under argon. The reaction mixture was allowed to stir at ambient temperature overnight. Solvents were removed from the reaction mixture and residue was extracted into EtOAC (200 mL), washed with NaHCO3 solution followed by standard workup. The desired product 218 was purified by flash silica gel column chromatography, eluent: hexane/EtOAc 3:2, 8.62 g (28.0%, bis DMT derivative) and 3-4% MeOH in chloroform, 15.28 g (57.3%, desired mono DMT derivative 218). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.63-7.60 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D2O), 7.38-7.17 (m, 15H, accounted for 14H after D2O exchange), 6.87-6.84 (d, J(H,H)=8.79 Hz, 4H), 4.98 (s, 2H), 4.62-4.59 (t, J(H,H)=5.37 Hz, 1H, exchangeable with D2O), 4.00-3.95 (m, 1H), 3.72 (s, 6H), 3.46-3.41 (t, J(H,H)=5.37 Hz, 2H), 3.00-2.87 (m, 4H), 2.08-2.04 (t, J(H,H)=7.33 Hz, 2H) 1.50-1.33 (m, 4H), 1.25-1.16 (m, 2H).
Compound 219:
Compound 218 (12.91 g, 20.16 mmol) in anhydrous pyridine (50 mL) was stirred with TBDMS-C1 (4.60 g, 30.52 mmol) in the presence of imidazole (6.30 g, 92.54 mmol) at ambient temperature for 6 h. After 6 h pyridine was removed in vacuo and the product was extracted into ethyl acetate (100 mL), washed with sodium bicarbonate solution followed by standard workup. The residue was purified by flash silica gel column chromatography, eluent: 2-3% methanol in dichloromethane to afford compound 219 (15.10 g, 99.3%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.65-7.63 (bd, J(H,H)=8.30 Hz, 1H, exchangeable with D2O); 7.38-7.17 (m, 14H); 6.86-6.84 (d, J(H,H)=8.79 Hz, 4H); 5.00 (s, 2H); 4.01-3.96 (m, 1H); 3.71 (s, 6H); 3.58-3.52 (m, 2H), 3.04-2.99 (m, 1H); 2.98-2.89 (m, 3H); 2.09-2.05 (t, 2H); 1.50-1.43 (m, 2H); 1.42-1.38 (m, 2H); 124-1.19 (m, 2H); 0.75 (s, 9H); −0.05 (s, 3H); −0.06 (s, 3H).
Compound 220:
Compound 219 (7.05 g, 9.33 mmol) and ammonium formate (3.00 g, 47.573) were suspended in 40 mL of methanol/ethyl acetate (1:2) and to this Pd—C (10%, 0.70 g) was added at ambient temperature. The suspension was initially warmed by blowing hot air and subsequently stirred at ambient temperature for 4 h. Completion of the reaction was monitored by TLC and after 4 h, the reaction mixture was filtered over a celite column, washed residue with methanol/ethyl acetate (1:2), combined filtrate and evaporated to dryness. Residue obtained was extracted into ethyl acetate (100 mL) and washed with aqueous sodium bicarbonate and water. Organic layer was dried over anhydrous Na2SO4 and evaporate to obtain the free amine. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.72-7.61 (m, 1.5H); 7.43-7.06 (m, 10H); 6.86-6.84 (d, J(H,H)=8.79 Hz, 4H); 4.20-3.97 (bm, 1H); 3.71 (s, 6H); 3.59-3.52 (bm, 2H); 3.07-3.00 (m, 2H); 2.93-2.88 (m, 2H); 2.72-2.98 (t, 1H); 2.10-2.05 (m, 2H); 1.53-1.44 (m, 4H), 1.28-1.22 (m, 2H), 0.75 (s, 9H); −0.05 (s, 3H); −0.07 (s, 3H).
The free amine (2.0 g, 3.22 mmol) was stirred with biotin-NHS ester (1.0 g, 2.93 mmol, purchased from Sigma) in the presence of triethylamine in DMF for 4 h. Progress of the reaction was monitored by TLC. Removed DMF in vacuo and the product was extracted into ethyl acetate (50 mL) and washed with water followed by standard workup. Compound 220 was purified by flash silica gel column chromatography; eluent: 5% methanol in dichloromethane, yield: 1.43 g (57.7%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.73-7.70 (t, J(H,H)=5.38 Hz, 1H, exchangeable with D2O); 7.66-7.63 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D2O); 7.38-7.18 (m, 9H); 6.86-6.84 (d, J(H,H)=8.30 Hz, 4H); 6.42-6.35 (d, J(H,H)=27.35 Hz, 2H, changes to 6 5.62-5.61 with J(H,H)=0.98 Hz after D2O exchange); 4.29-4.26 (m, 1H), 4.12-4.08 (m, 1H), 4.00-3.98 (m, 1H), 3.72 (s, 6H), 3.57-3.53 (m, 2H), 3.08-2.77 (m, 6H); 2.57-2.54 (d, J(H,H)=12.70 Hz, 1H), 2.10-2.06 (t, J(H,H)=8.79, 5.86 Hz, 2H); 2.04-2.00 (t, J(H,H)=7.32 Hz, 2H), 1.61-1.19 (m, 12H); 0.75 (s, 9H), −0.05 (s, 3H); −0.0.06 (s, 3H).
Compound 221:
Compound 220 (1.3 g, 1.54 mmol) was stirred with 4-tert-butylbenzoyl chloride (1 mL, 5.08 mmol) in the presence of DMAP (0.02 g, 0.163 mmol) in anhydrous pyridine (5 mL) under argon atmosphere for 4 h. Excess of 4-tert-butylbenzoyl chloride was quenched by adding methanol and subsequently removed pyridine and methanol in vacuo. The product was extracted into ethyl acetate (50 mL) and washed with aqueous sodium bicarbonate followed by standard workup. Residue obtained was subjected to flash column chromatography to afford compound 221 (0.64 g, 41.4%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.72-7.69 (t, J(H,H)=5.37 Hz, 1H, exchangeable with D2O); 7.64-7.62 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D2O); 7.53-7.17 (m, 14H); 6.83-6.81 (d, J(H,H)=8.79 Hz, 4H); 5.11-5.10 (d, J(H,H)=1.95 Hz, 2H); 4.01-3.98 (m, 1H); 3.71 (s, 6H); 3.61-3.55 (m, 3H); 3.18-3.15 (m, 1H); 3.03-2.88 (m, 4H); 2.07-1.99 (m, 4H); 1.62-1.58 (m, 1H); 1.47-1.19 (m, 22H); 0.75 (s, 9H), −0.05 (s, 3H); −0.07 (s, 3H).
13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 173.0, 172.9, 172.8, 172.7, 170.5, 170.0, 158.4, 155.5, 155.4, 152.2, 145.4, 136.2, 136.1, 132.0, 131.6, 130.2, 129.3, 129.1, 128.2, 128.1, 127.1, 125.2, 113.5, 85.6, 62.4, 62.2, 60.0, 59.9, 55.5, 55.2, 54.2, 50.8, 35.8, 35.1, 31.2, 31.1, 29.2, 29.0, 28.7, 26.4, 26.1, 25.6, 18.2, −5.1, −5.0.
Compound 222:
A solution of compound 221 (0.64 g, 0.64 mmol) in anhydrous THF (5 mL) was stirred with TEA.3HF (purchased from Aldrich, 1 mL) in the presence of anhydrous TEA (5 mL) at ambient temperature for 24. Solvents were removed from the reaction mixture under vacuum and the product was extracted into ethyl acetate, washed with aqueous sodium bicarbonate followed by standard workup. Flash silica gel column chromatography (eluant: 5% methanol in dichloromethane) of the residue afforded compound 222 (0.54 g, 95%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 7.91 (bs, 1H, exchangeable with D2O); 7.73 (bt, 1H, exchangeable with D2O); 7.63-7.61 (d, J(H,H)=8.79 Hz, 1H, exchangeable with D2O); 7.39-7.19 (m, 13H); 6.87-6.84 (d, J(H,H)=8.79 Hz, 4H); 5.05-5.02 (m, 1H), 4.62-4.60 (t, J(H,H)=5.37 Hz, 1H, exchangeable with D2O); 4.21-4.17 (m, 1H); 4.00-3.96 (m, 1H); 3.72 (s, 6H); 3.45-3.42 (t, J(H,H)=5.37 Hz, 2H, changed to a doublet after D2O exchange); 3.28-3.22 (m, 1H); 3.04-2.84 (m, 6H), 2.10-1.94 (m, 4H); 1.71-1.62 (m, 1H); 1.57-1.18 (m, 22H).
13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 172.8, 172.7, 169.5, 158.3, 155.8, 154.2, 145.4, 136.2, 132.8, 130.1, 128.8, 128.1, 128.0, 127.0, 124.6, 113.4, 85.4, 63.0, 62.1, 61.0, 57.6, 55.4, 55.2, 55.1, 51.1, 57.6, 55.4, 55.2, 55.1, 38.7, 37.7, 35.8, 35.6, 35.0, 31.3, 29.2, 28.6, 28.2, 26.4, 25.6, 25.5.
Compound 223:
The phosphoramidite 223 is prepared from compound 222 by reacting with 2-Cyanoethyl-N,N, N′, N′-tetraisopropylphosphorodiamidite in the presence of tetrazolediisopropylammonium salt in acetonitrile according to reported procedures (Rajeev et al., Org. Lett., 2003, 5, 3005).
Compound 224:
Compound 222 (0.50 g, 0.56 mmol) was stirred with succinic anhydride (0.115 g, 1.15 mmol) in the presence of DMAP (0.21 g, 1.72 mmol) in anhydrous ethylene dichloride under argon at 55° C. for 3 h. The reaction mixture was diluted to 20 mL by adding dichloromethane and washed with cold 10% citric acid solution, dried over anhydrous sodium sulfate and evaporated to dryness. The acid formed was purified by flash silica gel column chromatography (eluent: 10% methanol in dichloromethane); yield: 0.50 g (89.9%). 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 12.12 (s, 1H, exchangeable with D2O); 7.91 (bs, 1H, exchangeable with D2O); 7.86-7.84 (bd, 1H, exchangeable with D2O); 7.74-7.72 (bd, 1H, exchangeable with D2O); 7.52-7.16 (m, 13H); 6.88-6.86 (d, J(H,H)=8.79 Hz, 4H); 5.06-5.02 (m, 1H), 5.02-4.95 (m, 0.4H); 4.82-4.76 (m, 0.6H); 4.27-3.89 (m, 5H); 3.79-3.76 (m, 0.4H); 3.72 (s, 6H); 3.54-3.48 (m, 0.6H); 3.41-2.81 (m, 9H, accounted for 8H after D2O exchange); 2.52 (bm, 4H); 2.07-1.94 (bm, 4H); 1.71-1.62 (m, 1H); 1.54-1.13 (m, 22H).
13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 174.5, 174.1, 173.3, 173.2, 172.9, 172.7, 169.8, 158.6, 156.1, 155.9, 154.5, 145.3, 136.0, 132.9, 130.2, 129.3, 129.0, 128.5, 128.1, 127.4, 125.4, 124.8, 113.7, 85.8, 63.5, 62.3, 57.8, 55.6, 55.4, 55.2, 57.8, 55.6, 55.4, 55.2, 48.0, 37.9, 35.8, 35.2, 31.4, 31.4, 29.2, 29.1, 29.0, 28.8, 28.4, 26.4, 25.8, 25.6.
Compound 224 (2.0 with 56.15 μM/g loading) was prepared from the acid (0.45 g, 0.45 mmol), DMAP (0.068 g, 0.56 mmol), triphenylphosphine (0.135 mg, 0.51 mmol) and 2,2′-Dithiobis(5-nitropyridine) (DTNP, 0.16 g, 0.52 mmol) as described for the synthesis of compound 208a.
Compound 225a:
Compound 4a (12.09 g, 18.23 mmol) was stirred with TBDMS-Cl (4.00 g, 26.54 mmol) in the presence of imidazole (5.42 g, 79.61 mmol) in anhydrous pyridine (60 mm) overnight. After removing pyridine, the product was extracted into ethyl acetate (150 mL), washed with aqueous sodium bicarbonate, followed by standard workup. Residue obtained was subjected to flash silica gel column chromatography using 1% methanol in dichloromethane as eluent to afford compound 225a. 1H NMR (500 MHz, [D6]DMSO, 25° C.): δ 7.33-7.13 (bm, 15H, accounted for 14H after D2O exchange); 6.87-6.82 (bm, 4H); 5.01 (s, 0.2H, rotamer minor); 4.99 (s, 1.8H, rotamer major), 4.68-4.64 (m, 0.72H, major rotamer); 4.14-4.07 (bm, 1H), 3.72 (s, 7H), 3.38-3.36 (m, 0.6H, rotamer minor); 3.26-3.21 (m, 1.4H, rotamer major); 3.08-3.07 (m, 0.3H, rotamer, minor); 2.99-2.89 (m, 2.7H, rotamer, major); 2.22-2.12 (m, 2H), 2.04-1.78 (m, 2H); 1.48-1.23 (m, 6H), 0.84, 0.82 (s, 9H, rotamers major and minor); 0.05 (d, J(H,H)=1.5 Hz, 4.3H, rotamer major); 0.03-0.02 (d, J(H,H)=5.5 Hz, 1.7H).
Compound 226a
Compound 225a (12.22 g, 15.67 mmol) was hydrogenated at 1 atm over 10% Pd—C (1.12 g, wet Degussa type E101 NE/W) in ethyl acetate/methanol (4:1) for 4 h as described for the synthesis of compound 210a. The free amine obtained was stirred with biotin-NHS ester (5.42 g, 15.87 mmol, purchased from ChemGenes Corporation Wilmington, Mass.) in the presence of TEA for 6 h in dichloromethane/methanol (9:1, 100 mL). Solvents were removed from the reaction mixture and the product was extracted into dichloromethane (400 mL), washed with aqueous sodium bicarbonate followed by standard workup. The crude product thus obtained was used for next reaction without further purification or characterization.
Compound 227a:
The crude 226a and DMAP (0.31 g, 2.57 mmol) were taken in anhydrous pyridine and stirred over an ice bath. To the stirring solution, 4-tert-butylbenzoyl chloride (5.0 mL, 25.42 mmol) was added drop-wise over ten minute. After the addition, the reaction mixture was brought to room temperature over 2 h and continued stirring overnight. After quenching excess 4-tert-butylbenzoyl chloride by adding methanol, solvents were removed from the reaction mixture and the product was extracted into ethyl acetate (300 mL), washed with aqueous sodium bicarbonate followed by standard workup. The desired product 227a was purified by flash silica gel column chromatography using dichloromethane containing 4-6% of methanol as eluent. Yield: 9.53 g (58.9%). 1H NMR (500 MHz, [D6]DMSO, 25° C.): δ 7.90 (m, 1H, exchangeable with D2O); 7.73-7.72 (bm, 1H, exchangeable with D2O); 7.39 (s, 4H); 7.32-7.16 (m, 9H), 6.88-6.86 (m, 4H); 5.05-5.02 (m, 1H); 4.68-4.64 (m, 0.7H, rotamer, major); 4.57-4.53 (m, 0.3H, rotamer, minor); 4.20-4.17 (m, 1H), 4.13-4.08 (m, 1H); 3.72 (s, 6H), 3.38-3.20 (m, 3H); 3.08-2.81 (m, 5H); 2.24-1.77 (m, 6H); 1.68-0.98 (m, 23H); 0.84-0.81 (m, 9H), 0.05-0.02 (m, 6H).
Compound 228a
Compound 227a (6.43 g, 6. 22 mmol) was taken in a 250 ml RB and to this 3 mL of anhydrous TEA and 20 mL of 1M TBAF in anhydrous THF (purchased from Aldrich) were added under argon and stirred at ambient temperature for 4 h. Progress of the reaction was monitored by TLC, and after 4 h, THF was removed in vacuo. Residue was extracted into ethyl acetate (100 mL), washed with aqueous sodium bicarbonate followed by standard workup. Compound 228a was obtained as a white foamy solid after flash silica gel column chromatography (eluent: 5-6% methanol in dichloromethane), yield: 5.51 g (96. 3%). 1H NMR (500 MHz, [D6]DMSO, 25° C.): δ 7.90 (bs, 1H, exchangeable with D2O); 7.76-7.74 (m, 1H, exchangeable with D2O); 7.39 (s, 4H), 7.32-7.15 (m, 9H); 6.88-6.84 (m, 4H); 5.08-5.01 (m, 1H), 4.98-4.97 (d, 0.7H, exchangeable with D2O); 4.89-4.88 (d, 0.3H, exchangeable with D2O), 4.40-4.38 (m, 0.85H), 4.30-4.27 (m, 0.3H); 4.21-4.18 (m, 0.85H); 4.17-4.05 (m, 1H); 3.72 (s, 6H); 3.58-3.52 (m, 0.85H), 3.44-3.38 (m, 0.5H); 3.34-3.28 (m, 0.85H); 3.27-3.22 (m, 1H), 3.19-3.14 (m, 0.8H); 3.04-2.81 (m, 5H), 2.21-1.77 (m, 6H), 1.68-1.20 (m, 22H).
Compound 229a:
After drying over P2O5 under vacuum, compound 228a (1.49 g, 1.62 mmol) was taken in anhydrous dichloroethane (10 mL) under argon and stirred at ambient temperature. To the solution anhydrous TEA (0.70 mL, 5.02 mmol) and N,N-diisopropylamino 3-cyanoethylphosphonamidic chloride (0.80 mL, 3.38 mmol, purchased from ChemGenes Corporation, Wilmington, Mass.) were added and stirred for 3 h. After completion of the reaction, solvent and excess TEA were removed under vacuum and the product was extracted into ethyl acetate, washed with aqueous sodium bicarbonate solution followed by standard workup. Phosphoramidite 229a was purified by flash silica gel column chromatography using ethyl acetate as eluent, yield: 0.48 g (26.4%). 31P NMR (162 MHz, [D6]DMSO, 25° C.): δ 149.02 (major); 148.90 (minor); 148.62 (minor); 148.02 (major).
Compound 230a:
Compound 228a (1.55 g, 1.68 mmol) and DMAP (1.0 g, 8.18 mmol) were taken in anhydrous dichloroethane (5 mL) and stirred at ambient temperature. Succinic anhydride (0.30 g, 2.99 mmol) was added into the stirring solution and the stirring was continued overnight. The succinate derivative was obtained as a gray white solid (0.61 g, 35.5%) after workup and purification as described for the preparation of compound 224. 1H NMR (500 MHz, [D6]DMSO, 25° C.): δ 12.15 (s, 1H, exchangeable with D2O); 7.75-7.58 (bm, 1.4H, exchangeable with D2O); 7.54-7.47 (m, 4H); 7.32-7.16 (m, 9H); 6.89-6.81 (m, 4H); 5.38-5.33 (m, 0.7H); 5.28-5.20 (m, 0.3H); 5.06-5.00 (m, 0.3H); 4.99-4.95 (m, 0.7H); 4.82-4.77 (m, 1H); 4.28-4.22 (m, 0.25H); 4.21-4.15 (m, 0.75H); 3.80-3.60 (m, 7H), 3.57-3.42 (m, 2H); 3.22-2.80 (m, 7H); 2.28-1.90 (m, 6H), 1.58-1.41 (m, 24H).
13C NMR (100 MHz, [D6]DMSO, 25° C.): δ 174.3, 174.2, 173.2, 173.1, 172.7, 171.8, 170.3, 158.7, 158.5, 158.4, 155.9, 152.5, 145.4, 145.1, 140.6, 136.3, 136.1, 135.9, 131.9, 130.2, 129.4, 129.2, 128.4, 128.1, 127.3, 125.3, 113.8, 113.7, 113.3, 85.9, 73.4, 72.5, 63.6, 59.9, 55.6, 55.2, 52.8, 5.9, 35.8, 35.3, 35.0, 34.6, 33.5, 31.6, 31.4, 31.3, 29.3, 29.1, 28.6, 28.5, 28.1, 26.5, 25.5, 24.6.
The solid support 230a (2.05 g with a loading of 66.88 μM/g) was obtained from the succinate (0.57 g, 0.55 mmol), DMAP (0.085 g, 0.69 mmol), triphenylphosphine (0.15 g, 0.57 mmol), DTNP (0.18 g, 0.58 mmol) and lca-CPG (155 M/g loading with a mean pore diameter of 484 Å, purchased from Millipore) as described for the preparation of compound 208a.
Compound 225b:
Compound 225b is prepared from compound 4b as described for the synthesis of compound 225a.
Compound 226b:
The desired compound is obtained from compound 225b as described for the preparation of compound 226a from compound 225a.
Compound 227b:
The desired compound is obtained from compound 226b as described for the preparation of compound 227a from compound 226a.
Compound 228b:
Compound 228b is prepared from compound 227b as described for the synthesis of compound 228a from compound 227a.
Compound 229b:
The desired compound is obtained from compound 228b as described for the preparation of compound 229a from compound 228a.
Compound 230b:
Compound 230b is prepared from compound 228b as described for the synthesis of compound 230a from compound 228a.
Compound 232a:
Tert-Butyl ester (231a) of 6-hydroxyhexanoic acid is prepared as reported in the literature (Larock and Leach, J. Org. Chem, 1984, 49, 2144). Compound 231a is reacted with N-hydroxyphthalimide under Mitsunobu conditions to obtain compound 232a (as reported by Katajisto et. al., Bioconjugate Chem., 2004, 15, 890.
Compound 233a:
Compound 232a is treated with trifluoroacetic acid to obtain compound 233a.
Compound 234a:
The free acid 233a is stirred with N-hydroxysuccinimide and DCC in the presence of DAMP in DMF for 30 min and subsequently compound 38 is added into the reaction mixture to obtain the desired compound 234a.
Compound 235a:
Compound 234a is treated with hydrazine.hydrate in pyridine and subsequently with 5-cholesten-3-one (purchased from Aldrich) to obtain compound 235a.
Compound 236a:
The phosphoramidite 236a is prepared from 235a as described for the compound 7 from compound 6 using 2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as the phosphitylation agent.
Compound 237a:
The solid support 237a is obtained from compound 235a as described for the synthesis of compound 208a.
Compound 238a:
Compound 235a is treated with sodium cyanoborohydride to reduce the C═N double bond. The crude product of the sodium cyanoborohydride reaction is subsequently treated with ethyl trifluoroacetate in the presence of TEA in dichloromethane to obtain compound 238a.
Compound 239a:
The phosphoramidite 239a is prepared from 238a as described for the compound 7 from compound 6 using 2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as the phosphitylation agent.
Compound 240a:
The solid support 240a is obtained from compound 238a as described for the synthesis of compound 208a.
Compound 232b:
Compound 231b is prepared as reported in the literature by Noguchi et al. (Tetrahedron, 1995, 51, 10531). Compound 231b is converted to 232b as described for the preparation of compound 232a.
Compound 236b:
The desired phosphoramidite is obtained from compound 232b in four steps as described for the preparation of compound 236a from 232a.
Compound 237b:
The CPG support is obtained from compound 235b as described for the synthesis of compound 208a from 207a.
Compound 238b:
Compound 238b is prepared from compound 235b as described for the preparation of compound 238a from 235a.
Compound 239b:
The phosphoramidite 239b is prepared from 238b as described for the compound 7 from compound 6 using 2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as the phosphitylation agent.
Compound 240b:
The solid support 240b is obtained from compound 238b as described for the synthesis of compound 208a.
Compound 241a:
Naproxen pentafluorophenol ester (1.3 g, 3.28 mmol) was added into a solution of compound 213 (1.5 g, 2.14 mmol, purchased from ChemGenes Corporation, Wilmington, Mass.) and TEA (4.6 mL, 33.0 mmol) and stirred overnight. Solvent and excess TEA were removed from the reaction in vacuo and the product was extracted into ethyl acetate (80 mL), washed with aqueous sodium bicarbonate solution followed by standard workup. Flash silica gel column chromatography of the residue using dichloromethane containing 4% methanol as eluent yielded 0.85 g (43.5%) of compound 241a as a grayish white solid. 1H NMR (400 MHz, [D6]DMSO, 25° C.): δ 11.62 (s, 1H, exchangeable with D2O); 7.99-7.91 (bm, 3H), 7.76-7.68 (m, 3H); 7.43-7.01 (m, 13H); 6.87-6.84 (m, 4H); 6.17-6.14 (t, J′(H,H)=6.41 and J″(H,H)=6.71 Hz, 1H), 5.28-5.27 (d, J(H,H)=4.88 Hz, 1H, exchangeable with D2O); 4.23-4.19 (m, 1H); 3.88-3.81 (bm, 4H), 3.69-3.68 (bm, 8H); 3.20-2.97 (m, 6H); 2.34-2.27 (m, 1H); 2.19-2.13 (m, 1H); 1.38-1.17 (bm, 11H).
Compound 242a:
Compound 241a (0.65 g, 0.71 mmol) and DMAP (0.13 g, 1.06 mmol) were taken in dichloroethane (5 mL) in an RB and stirred at ambient temperature. Succinic anhydride (0.11 g, 1.09 mmol) was added into the stirring solution and the stirring was continued for 24 h. Progress of the reaction was monitored by TLC and after 24 h, the reaction mixture was diluted to 50 mL by adding ethyl acetate. Organic layer was washed with cold dilute citric acid solution followed by water. Organic layer was dried over anhydrous sodium sulfate and evaporated in vacuo to obtain the corresponding succinic acid derivative (0.67 g, 92.8%, crude yield) of compound 241a.
The succinate (0.51 g, 0.50 mmol) thus obtained was converted to compound 242a (2.8 g, with a loading of 11.6 μM/g) by coupling to lca-CPG (2.8 g with initial loading of 112.7 μM/g with mean pore size 505 A, purchased from Millipore) using triphenylphosphine (0.132 g, 0.503 mmol), DAMP (0.07 g, 0.57 mmol) and 2,2′-dithiobis(5-nitropyridine) (DTNP, 0.156 g, 0.50 mmol) as coupling agents as described for the synthesis of compound 208a.
Compound 243a:
The phosphoramidite 243a is prepared from 241a as described for the compound 7 from compound 6 using 2-cyanoethyl-N,N,N′,N″-tetraisopropylphosphorodiamidite as the phosphitylation agent.
Example 16
Oligonucleotide Synthesis, Purification and Analysis
Synthesis:
The Oligonucleotide molecules were synthesized on a 394 ABI machine using the standard 93 step cycle written by the manufacturer with modifications to a few wait steps as described below. The solid support was available in house and the monomers were RNA phosphoramidites with fast protecting groups (5′-O-dimethoxytrityl N6-phenoxyacetyl-2′-O-t-butyldimethylsilyladenosine-3′-O—N,N′-diisopropyl-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-O-t-butyldimethyl silylcytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-p-isopropylphenoxyacetyl-2′-O-t-butyldimethyl silylguanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-O-t-butyldimethylsilyluridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite from Pierce Nucleic Acids Technologies. All 2′-O-Me amidites were received from Glen Research. All amidites were used at a concentration of 0.15M in acetonitrile (CH3CN) and a coupling time of 12-15 min. The activator was 5-(ethylthio)-1H-tetrazole (0.25M), for the PO-oxidation Iodine/Water/Pyridine was used and for PS-oxidation, 2% Beaucage reagent (Iyer et al., J. Am. Chem. Soc., 1990, 112, 1253) in anhydrous acetonitrile was used. The sulphurization time was about 6 min.
Deprotection-I (Nucleobase Deprotection)
After completion of synthesis the support was transferred to a screw cap vial (VWR Cat #20170-229) or screw caps RNase free microfuge tube. The oligonucleotide was cleaved from the support with simultaneous deprotection of base and phosphate groups with 1.0 mL of a mixture of ethanolic ammonia [ammonia: ethanol (3:1)] for 15h at 55° C. The vial was cooled briefly on ice and then the ethanolic ammonia mixture was transferred to a new microfuge tube. The CPG was washed with 2×0.1 mL portions of RNase free deionised water. Combined washings, cooled over a dry ice bath for 10 min and subsequently dried in speed vac.
Deprotection-II for RNA Oligonucleotides(Removal of 2′ TBDMS Group)
The white residue obtained was resuspended in 400 μl of triethylamine, triethylamine trihydrofluoride (TEA.3HF) and NMP (4:3:7) and heated at 50° C. for overnight to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′position (Wincott et al., Nucleic Acids Res., 1995, 23, 2677). The reaction was then quenched with 400 μl of isopropoxytrimethylsilane (iPrOMe3Si, purchased from Aldrich) and further incubated on the heating block leaving the caps open for 10 min; (This causes the volatile isopropxytrimethylsilylfluoride adduct to vaporize). The residual quenching reagent was removed by drying in a speed vac. Added 1.5 ml of 3% triethylamine in diethyl ether and pelleted by centrifuging. The supernatant was pipetted out without disturbing the pellet and the pellet was dried in speed vac. The crude RNA was obtained as a white fluffy material in the microfuge tube.
Quantitation of Crude Oligomer or Raw Analysis
Samples were dissolved in RNase free deionied water (1.0 mL) and quantitated as follows: Blanking was first performed with water alone (1 mL) 20 μL of sample and 980 μL of water were mixed well in a microfuge tube, transferred to cuvette and absorbance reading obtained at 260 nm. The crude material is dried down and stored at −20° C.
5. Purification of Oligomers:
PAGE Purification
PAGE purification of oligomers synthesized was performed as reported by Sambrook et al. (Molecular Cloning: a Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The 12% denaturing gel was prepared for purification of unmodified and modified oligoribonucleotides. Took 120 mL Concentrate+105 mL Diluents+25 mL Buffer (National Diagnostics) then added 50 μL TEMED and 1.5 mL 10% APS. Pour the gel and leave it for ½ h to polymerize. Suspended the RNA in 20 μL water and 80 μL formamide. Load the gel tracking dye on left lane followed by the sample slowly on to the gel. Run the gel on 1×TBE buffer at 36 W for 4-6h. Once run is completed, Transfer the gel on to preparative TLC plates and see under UV light. Cut the bands. Soak and crushed in Water. Leave in shaker for overnight. Remove the eluent, Dry in speed vac.
HPLC Analysis and Purification
Analysis was performed on an Agilent 1100 series HPLC using a Dionex 4×250 mm DNAPak column. Buffer A was 1 mM EDTA, 25 mM Tris pH9, 50 mM NaClO4, 20% MeCN. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO4, 20% MeCN. Separation was performed on a 0-65% B segmented gradient with buffers and column heated to 65° C.
Materials were purified on an ÅKTA Explorer equipped with a column packed with TSKgel Q 5PW (Tosoh Biosciences). Buffer A was 1 mM EDTA, 25 mM Tris pH 9. Buffer B was 1 mM EDTA, 25 mM Tris pH 9, 0.4 M NaClO4. Buffers were heated by a 4 kW buffer heater set at 65° C., giving a column outlet temperature of 45° C. The solution containing the crude material was diluted 4-6 fold and loaded onto the column and eluted with a segmented gradient from 0-60% B. Appropriate fractions were pooled
Desalting of Purified Oligomer
The purified dry oligomer was then desalted using Sephadex G-25 M (Amersham Biosciences). The cartridge was conditioned with 10 mL of RNase free deionised water thrice. Finally the purified oligomer was dissolved in 2.5 mL RNasefree water and passed through the cartridge with very slow drop wise elution. The salt free oligomer was eluted with 3.5 mL of RNase free water directly into a screw cap vial. All oligonucleotides were finally analyzed by LC-MS and capillary gel electrophoresis.
TABLE 6
List of ligand oligonucleotides (sense and antisense strand).
Found
Cal Mass
Mass
CGE
Sequence ID
Sequence
amu
amu
(%)
100
5′ CUU ACG CUG AGU ACU UCG A dTdT 3′
6606.00
6606.45
99.25
101
5′ UCG AAG UAC UCA GCG UAA G dT dT 3′
6696.32
6693.0
89.0
102
5′ CUU ACG CUG AGU ACU UCG A dTdT L13′
7084.19
7084.58
96.90
103
5′ UCG AAG UAC UCA GCG UAA G dT dT L1 3′
7170.29
7170.89
92.00
104
5′ CUU ACG CUG AGU ACU UCG A dT dT* L1 3′
7100.19
7099.12
92.99
105
5′ UCG AAG UAC UCA GCG UAA G dT dT* L1 3′
7157.29
7156.2
89.00
106
5′G*G*U*G*U*A U G G C U U C A A C C* C* U* U*2′OMe
7293.00
7237.06
97.50
U*2′OMe L1 3′
107
5′A* G* G* G* U U G A A G C C A U* A C * A* C* C* U*2′OMe
7362.73
7338.4
96.00
U2′OMe L1 3′
108
5′ CUU ACG CUG AGU ACU UCG A dT dT L2 3′
7064.96
7064.91
90.0%
109
5′ UCG AAG UAC UCA GCG UAA G dT dT L23′
7154.00
7153.2
90.72
110
5′ UCG AAG UAC UCA GCG UAA G UU L2 3′
7153.98
7151.13
92.20
111
5′ L1CUU ACG CUG AGU ACU UCG A dTdT 3′
112
5′ L1UCG AAG UAC UCA GCG UAA G dT dT 3′
113
5′ L2CUU ACG CUG AGU ACU UCG A dTdT 3′
114
5′ L2UCG AAG UAC UCA GCG UAA G dT dT 3′
115
5′ CUU ACG CUG AGU ACU UCG A dTdTL3 3′
116
5′ UCG AAG UAC UCA GCG UAA G dT dTL3 3′
117
5′ L3CUU ACG CUG AGU ACU UCG A dTdT 3′
118
5′ L3 UCG AAG UAC UCA GCG UAA G dT dT 3′
119
5′ CUU ACG CUG AGU ACU UCG A dTdT L43′
120
5′ UCG AAG UAC UCA GCG UAA G dT dT L4 3′
121
5′ L4CUU ACG CUG AGU ACU UCG A dTdT 3′
122
5′ L4 UCG AAG UAC UCA GCG UAA G dT dT 3′
123
5′ CUU ACG CUG AGU ACU UCG A dT dT L53′
124
5′ UCG AAG UAC UCA GCG UAA G dT dT L5 3′
125
5′ L5CUU ACG CUG AGU ACU UCG A dT dT 3′
126
5′ L5UCG AAG UAC UCA GCG UAA G dT dT 3′
127
5′ CUU ACG CUG AGU ACU UCG A dT dT L63′
128
5′ UCG AAG UAC UCA GCG UAA G dT dT L6 3′
129
5′ L6CUU ACG CUG AGU ACU UCG A dT dT 3′
130
5′ L6UCG AAG UAC UCA GCG UAA G dT dT 3′
131
5′ CUU ACG CUG AGU ACU UCG A dT dT L7 3′
132
5′ UCG AAG UAC UCA GCG UAA G dT dT L73′
133
5′ L7CUU ACG CUG AGU ACU UCG A dT dT 3′
134
5′ L7UCG AAG UAC UCA GCG UAA G dT dT 3′
135
5′ CUU ACG CUG AGU ACU UCG A dT dT L8 3′
136
5′ UCG AAG UAC UCA GCG UAA G dT dT L83′
137
5′ L8CUU ACG CUG AGU ACU UCG A dT dT 3′
138
5′ L8UCG AAG UAC UCA GCG UAA G dT dT 3′
139
5′ CUU ACG CUG AGU ACU UCG A dT dT Ls L9 3′
140
5′ UCG AAG UAC UCA GCG UAA G dT dT L9 3′
141
5′ L9CUU ACG CUG AGU ACU UCG A dT dT 3′
142
5′ L9UCG AAG UAC UCA GCG UAA G dT dT 3′
143
5′ CUU ACG CUG AGU ACU UCG A dT dT L103′
144
5′ UCG AAG UAC UCA GCG UAA G dT dT L10 3′
145
5′ L10CUU ACG CUG AGU ACU UCG A dT dT 3′
146
5′ L10UCG AAG UAC UCA GCG UAA G dT dT 3′
147
5′ CUU ACG CUG AGU ACU UCG A dT dT L11 3′
148
5′ UCG AAG UAC UCA GCG UAA G dT dT L3′
149
5′ L11CUU ACG CUG AGU ACU UCG A dT dT 3′
150
5′ L11UCG AAG UAC UCA GCG UAA G dT dT 3′
151
5′ CUU ACG CUG AGU ACU UCG A dT dT L12 3′
152
5′ UCG AAG UAC UCA GCG UAA G dT dT L123′
153
5′ L12CUU ACG CUG AGU ACU UCG A dT dT 3′
154
5′ L12UCG AAG UAC UCA GCG UAA G dT dT 3′
155
5′ CUU ACG CUG AGU ACU UCG A dT dT L13 3′
156
5′ UCG AAG UAC UCA GCG UAA G dT dT L133′
157
5′ L13CUU ACG CUG AGU ACU UCG A dT dT 3′
158
5′ L13UCG AAG UAC UCA GCG UAA G dT dT 3′
159
5′ CUU ACG CUG AGU ACU UCG A dT dT L14 3′
160
5′ UCG AAG UAC UCA GCG UAA G dT dT L143′
161
5′ L14CUU ACG CUG AGU ACU UCG A dT dT 3′
162
5′ L14UCG AAG UAC UCA GCG UAA G dT dT 3′
163
5′ CUU ACG CUG AGU ACU UCG A dT dT L15 3′
164
5′ UCG AAG UAC UCA GCG UAA G dT dT L153′
165
5′ L15CUU ACG CUG AGU ACU UCG A dT dT 3′
166
5′ L15UCG AAG UAC UCA GCG UAA G dT dT 3′
167
5′ CUU ACG CUG AGU ACU UCG A dT dT L16 3′
168
5′ UCG AAG UAC UCA GCG UAA G dT dT L163′
169
5′ L16CUU ACG CUG AGU ACU UCG A dT dT 3′
170
5′ L16UCG AAG UAC UCA GCG UAA G dT dT 3′
171
5′ CUU ACG CUG AGU ACU UCG A dT dT L17 3′
172
5′ UCG AAG UAC UCA GCG UAA G dT dT L173′
173
5′ L17CUU ACG CUG AGU ACU UCG A dT dT 3′
174
5′ L17UCG AAG UAC UCA GCG UAA G dT dT 3′
L1 = Naproxen 6-aminohexanoic acid with Serinol linker
L2 = Ibuprofen 6-aminohexanoic acid with Serinol linker
L3 = Cholesterol 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L4 = Cholesterol 6-aminohexanoic acid with serinol linker
L6 = Cholesterol with trans-4-hydroxy-L-prolinol linker containing cationic tert-amine moiety
L7 = Thiocholesterol with trans-4-hydroxy-L-prolinol linker
L8 = Cholesterol 6-aminohexanoic acid with 3-hydroxy-4-(hydorxy)methylpyrrolidine linker
L8 = Biotin 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L9 = Biotin 6-aminohexanoic acid with serinol linker
L10 = Biotin 12-aminododecanoic acid with trans-4-hydroxy-L-prolinol linker
L10 = ω-aminocaproyl with trans-4-hydroxy-L-prolinol linker
L11 = ω-aminododecyl with trans-4-hydroxy-L-prolinol linker
L12 = Vitamin E 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L13 = Dialkylglyceride 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L14 = Naproxen 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L15 = N,N-Dimethyl 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
L16 = N,N-Dimethyl 12-aminododecanoic acid with trans-4-hydroxy-L-prolinol linker
L17 = Nadixic 6-aminohexanoic acid with trans-4-hydroxy-L-prolinol linker
* = PS
Example 17
siRNA Modifications Enhanced Duplex Stability
Radiolabel Method for Monitoring Serum Stability of siRNA Duplexes:
siRNA duplexes were prepared at a stock concentration of 1 μM in which either the sense (S) or antisense strand (AS) contained a trace amount of 5′-32P labeled material (e.g. 32P-S/AS and S/32P-AS). The presence of the end-labeled sense or antisense strand allowed for monitoring of the individual strand within the context of the siRNA duplex. Therefore, two duplex preparations were made for each siRNA sequence tested. siRNA duplexes were incubated in 90% human serum at a final concentration of 100 nM duplex. Samples were removed and quenched in a stop mix at appropriate times. For a typical time course, 10 seconds, 15 minutes, 30 minutes, 1 hour, 2 hours and 4 hours time points were taken. Samples were analyzed by denaturing polyacrylamide gel electrophoresis along with a control sample (4 hour buffer-alone incubation) and a partial alkaline hydrolysis ladder of the labeled sense or antisense strand as a marker. The gel was imaged using a Fuji phosphorimager to detect the full length sense and antisense strands along with any degradation fragments that were generated by serum nucleases during incubation.
Since there is the possibility of losing the 5′ phosphate label due to phosphatase activity in the serum, an alternative to 5′ end labeling is to place an internal 32P or 33P label within either the sense or antisense strand. This labeling method is much more laborious than 5′ end labeling and currently we have no evidence that dephosphorylation occurs during serum incubation.
A series of chemical modifications that fall into the following categories; backbone modification, sugar modification, nucleobase modification and 3′ conjugate, were tested and showed enhanced serum stability as compared to a unmodified siRNA duplex. A description of each modification, its location within the siRNA duplex, and the serum stability data follows.
Serum Stability of Unmodified Parent Duplex:
The unmodified parent duplex, AL-DUP-1000, was used to establish the serum stability baseline for evaluating the effect of chemical modifications on nuclease resistance.
AL-DUP-1000
ALN-SEQ-1000
SEQ ID NO: 54
5′-CUUACGCUGAGUACUUCGAdTdT-3′
ALN-SEQ-1001
SEQ ID NO: 61
3′dTdTGAAUGCGACUCAUGAAGCU-5′
AL-DUP-1000 was subjected to the serum stability assay to evaluate its inherent nuclease resistance and to define its degradation pattern (FIG. 21). Denaturing gel electrophoresis was used analyze AL-DUP-1000 in a human serum stability assay. An siRNA duplex containing 5′ end-labeled sense RNA (*s/as) and a duplex containing 5′ end-labeled antisense RNA (as/s*) were each incubated in 90% human serum and time points were assayed at 10 seconds, 5 min, 15 min, 30 min, 1 hour, 2 hours and 4 hours. The control was a 4 hour time point for siRNA duplex incubated in PBS buffer alone, OH— was the partial alkaline hydrolysis marker. This unmodified duplex was observed to be degraded by both 3′-5′ exonucleases and endonucleases (FIG. 21).
Cleavage of the 3′ end of both the sense and antisense strands by 3′-5′ exonucleases occurs within the first 5 minutes of incubation resulting in the loss of the 3′ terminal dT residues (top vertical lines in s*/as and s/as* panels of FIG. 21). In addition to exonuclease degradation, both strands were cleaved by endonucleases. There was a major endonuclease site at position sixteen of the antisense strand (bottom vertical lines in s*/as and s/as* panels of FIG. 21) that appears as early as 10 seconds. Very little full length sense or antisense strand was remaining after 1 hour in human serum. Chemical modifications were introduced within the context of the parent duplex to evaluate their effect on nuclease resistance. These chemical modifications fall within one of the following classes: backbone modification, sugar modification, nucleobase modification, cationic modification and conjugate.
Backbone Modifications Enhanced Nuclease Resistance:
Specific phophodiester linkages of the siRNA duplex were replaced by either phosphorothioate or methylphosphonate and their stability was evaluated in the human serum stability assay. Table 7 contains the sequences of the duplexes tested. Substitution of the phosphodiester linkage at the 3′ end of both the sense and antisense strands inhibited exonucleolytic degradation of the 3′ overhangs (FIGS. 22A and 22B) as compared to the unmodified parent duplex (refer to FIG. 21). Full length starting material was present for four hours for both the sense and antisense strands. The endonucleolytic cleavage pattern seen in the unmodified duplex was unchanged. Similar results were obtained for duplexes that contained additional phosphorothioates at their 3′ ends (data not shown). The placement of phosphorothioates at the endonucleolytic cleavage sites (duplexes 1419, 1420 and 1421) did not inhibit endonucleolytic cleavage at these sites (data not shown). In summary, a single phosphorothioate or methylphosphonate between the two 3′ terminal nucleotides was sufficient to protect the 3′ ends from exonuclease degradation. Additional phosphorothioates at the 3′ ends appear to enhance this effect, which may be necessary for long term exposure to serum nucleases.
TABLE 7
siRNA duplexes containing backbone modifications.
Alnylam
Alnylam Duplex
Duplex Sequence
Sequence
AL-DUP-1393
5′-CUUACGCUGAGUACUUCGAdT*dT-3′
AL-SEQ-1026
3′-dT*dTGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1027
AL-DUP-1394
5′-CUUACGCUGAGUACUUCGA*dT*dT-3′
AL-SEQ-1028
3′-dT*dT*GAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1029
AL-DUP-1395
5′-CUUACGCUGAGUACUUCG*A*dT*dT-3′
AL-SEQ-1030
3′-dT*dT*G*AAUGCGACUCAUGAAGCU-5′
AL-SEQ-1031
AL-DUP-1396
5′-CUUACGCUGAGUACUUC*G*A*dT*dT-3′
AL-SEQ-1032
3′-dT*dT*G*A*AUGCGACUCAUGAAGCU-5′
AL-SEQ-1033
AL-DUP-1419
5′-CUUACGCUGAGU*ACUUCGAdTdT-3′
AL-SEQ-2182
3′-dTdTGAAUGCGACUCA*UGAAGCU-5′
AL-SEQ-2184
AL-DUP-1420
5′-CUU*ACGCUGAGU*ACUUCGAdTdT-3′
AL-SEQ-2183
3′-dTdTGAA*UGCGACUCA*UGAAGCU-5′
AL-SEQ-2185
AL-DUP-1421
5′-CUU*ACGCUGAGU*ACUUCGAdT*dT-3′
AL-SEQ-2186
3′-dT*dTGAA*UGCGACUCA*UGAAGCU-5′
AL-SEQ-2188
AL-DUP-1329
5′-CUUACGCUGAGUACUUCGAdTmpdT-3′
AL-SEQ-1038
3′-drimpdTGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1039
(* = phosphorothioate, mp = methylphosphonate)
SEQ ID NOs 62-77, respectively.
Sugar Modifications Enhanced Nuclease Resistance:
The effect of replacing the 2′OH with 2′OMe was evaluated at the sites of endonucleolytic cleavage as well as at the 3′ ends of the siRNA duplex. The duplexes tested in the human serum stability assay are shown in Table 2. Some of these duplexes also contained phosphorothioate linkages to evaluate whether the combination of the two modifications enhance nuclease resistance more significantly. Substitution of the terminal dT residues with 2′OMe-U (AL-DUP-1027) reduced 3′-5′ exonuclease degradation slightly over the unmodified parent duplex (data not shown); however, the extent of exonuclease protection by 2′OMe-U was far less than that achieved by placing a
TABLE 8
siRNA duplexes containing 2′OMe Substitutions
Alnylam Duplex
Duplex Sequence
Alnylam Sequence
AL-DUP-1027
5′-CUUACGCUGAGUACUUCGAUU-3′
AL-SEQ-1006
3′-UUGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1007
AL-OUP-1036
5′-C*UUACGCUGAGUACUUCGAU*U-3′
AL-SEQ-1008
3′-U*UGAAUGCGACUCAUGAAGC*U-5′
AL-SEQ-1009
AL-DUP-13ff
5′-C*UUACGCUGAGUACUUCGAU*U,-3′
AL-SEQ-gggg
3′-U*UGAAUGCGACUCAUGAAGC*U-5′
AL-SEQ-hhhh
AL-DUP-1363
5′-C*UUACGCUGAGUACUUCGAU*U-3′
AL-SEQ-1162
3′-U*UGAAUGCGACUCAUGAAGC*U-5′
AL-SEQ-1163
(U = 2′OMe-uridine, * = phosphorothioate)
SEQ ID NOs 78-85, respectively.
phosphorothioate between the two terminal dT residues (see FIG. 22A). Addition of a single phosphorothioate between the two terminal 2′OMe-uridine residues effectively inhibited 3′-5′ exonucleolytic cleavage as seen in FIG. 23 for duplexes AL-DUP-1036, AL-DUP-13ff, and AL-DUP-1363. 2′OMe substitution on its own was much more effective at protecting from endonucleolytic cleavage when placed at the internal cleavage sites. The parent duplex was cleaved 3′ of U at two UpA sites within the duplex. Both strands are cleaved due to the symmetry of this dinucleotide repeat and mapping data was used to confirm the sites of cleavage (data not shown). Placement of 2′OMe at the strong endonucleolytic site ((FIG. 23, star in s/*as gel, AL-DUP-13ff) resulted in inhibition of cleavage at this site. The second, weaker endonucleolytic site (FIG. 23, black star in *s/as), however, was slightly enhanced when the strong site was protected with 2′OMe (FIG. 23, compare AL-DUP-13ff to AL-DUP-1036). Protection of both sites with 2′OMe (AL-DUP-1363) resulted in reduced endonucleolytic cleavage at both sites (FIG. 23). The inhibitory effect of the 2′OMe substitution is consistent with the mechanism of endonucleolytic cleavage, which requires the 2′OH as a nucleophile in the cleavage reaction. 2′OMe modification will also be an effective means to protect the 3′ overhang of single overhang siRNA duplexes where the 3′ overhang is composed of ribonucleotides. In this situation, 2′OMe substitution can be used to block the possible loss of the terminal two nucleotides by endonucleolytic cleavage and phosphorothioate can be used to protect from exonuclease degradation.
Cationic Modifications Enhanced Nuclease Resistance:
The effect of three different cationic chemical modifications on nuclease resistance was tested and compared to the parent unmodified duplex. The structures of the three cationic modifications tested are shown below.
TABLE 9
siRNA duplexes containing cationic substances
Alnylam
Alnylam Duplex
Duplex Sequence
Sequence
AL-DUP-10aa
5′-CUUACGCUGAGUACUUCGAdTaadT-3′
AL-SEQ-1017
3′-a adTdTGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1018
AL-DUP-10bb
5′-CUUACGCUGAGUACUUCGAaadTaadT-3′
AL-SEQ-1015
3′-aadTaadTGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-1016
AL-DUP-1ccc
5′-CUUACGCUGAGUACUUCGAdTdTAbP-3′
AL-SEQ-dddd
3′-AbPdTdTGAAUGCGACUCAUGAAGCU-5′
AL-SEQ-eeee
AL-DUP-1403
5′-C*UaaUACGCUGAGUACUUCGAU*U-3′
AL-SEQ-2080
3′-U*UGAAaaUGCGACUCAUGAAGC*U-5′
AL-SEQ-2081
AL-DUP-1406
5′-C*UaaUACGCUGAGaaUACUUCGAU*U-3′
AL-SEQ-2082
3′-U*UGAAaaUGCGACUCAaaUGAAGC*U-5′
AL-SEQ-2083
(aadT = alkylamine-dt, abP = abasic pyrrolidine cationic, aaU = allylamine-U, * = phosphorothioate, U = 2′OMe-U)
SEQ ID NOs 86-95, respectively.
The sequences of the duplexes assayed in the human serum stability assay are shown in Table 9. Both alkylamino-dT and abasic pyrrolidine cationic modifications were placed at the 3′ terminal overhang to evaluate their effect on 3′-5′ exonuclease degradation. Allylamino-uridines were placed at the internal endonucleolytic cleavage sites to evaluate their ability to inhibit endonucleolytic cleavage. As seen in FIG. 24, replacing the 3′ terminal dT residue with a single alkylamino-dT efficiently inhibited 3′-5′ exonucleolytic degradation (FIG. 24, AL-DUP-10aa, left gel image). Replacement of both dT residues in the overhang with alkylamino-dT resulted in a similar extent of inhibition (data not shown). Addition of an abasic pyrrolidine cationic modification at the 3′ terminus of each strand also protected against exonucleolytic degradation (FIG. 24, middle gel image). Both the alkylamino-dT and abasic pyrrolidine modifications protected from 3′-5′ exonucleolytic cleavage up to 23 hours (data not shown). Placement of allylamino-U at the internal cleavage sites inhibited endonucleolytic cleavage as shown in FIG. 24 for duplex AL-DUP-1403. The ends of this duplex were stabilized from exonucleolytic degradation by 2′OMe-U and phosphorothioate substitutions in order to separate the two different cleavage events. Endonucleolytic cleavage was inhibited at both internal cleavage sites by allylamino-U substitution for AL-DUP-1406 (data not shown).
3′ Conjugates Enhanced Nuclease Resistance:
Conjugation of naproxen and ibuprofen to the 3′ end of the siRNA were tested for their ability to inhibit 3′-5′ exonucleolytic degradation. The structure of naproxen is shown in below:
Table 10 lists the siRNAs that were tested in the human serum stability assay. Conjugation of either naproxen or ibuprofen to the 3′ end inhibited exonucleolytic degradation. FIG. 18 shows the serum stability data for the naproxen modified duplex (AL-DUP-1069) and similar results were obtained for AL-DUP1413. Presumably the conjugates inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3′ end of the siRNA duplex. Similar data was also obtained for AL-DUP-1069 in pooled mouse serum.
TABLE 10
siRNA duplexes containing 3′ conjugates
Alnylam
Alnylam
Duplex
Duplex Sequence
Sequence
AL-DUP-
5′-CUUACGCUGAGUACUUCGAdTdTNap-3′
1069
3′-NapdTdTGAAUGCGACUCAUGAAGCU-5′
AL-DUP-
5′-CUUACGCUGAGUACUUCGAdTdTIbu-3′
1413
3′-NapdTdTGAAUGCGACUCAUGAAGCU-5′
(Nap = Naproxen, Ibu = Ibuprofen)
SEQ ID NOs 96-99, respectively.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
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16523411
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alnylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 27th, 2022 09:15AM
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Apr 27th, 2022 09:15AM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Apr 19th, 2022 12:00AM
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Nov 22nd, 2021 12:00AM
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https://www.uspto.gov?id=US11306314-20220419
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Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof
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The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.
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11306314
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1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the nucleotide sequence of 5′-AAAGACUGAUCAAAUAUGUUGAG-3′ (nucleotides 274-296 of SEQ ID NO:1),
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the nucleotide sequence of 5′-AAGACUGAUCAAAUAUGUUGAGU-3′ (nucleotides 273-295 of SEQ ID NO:1),
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
3. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the nucleotide sequence of 5′-AGACUGAUCAAAUAUGUUG-3′ (nucleotides 276-294 of SEQ ID NO:1),
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
4. The dsRNA agent of claim 1, wherein each strand is independently 19-25 nucleotides in length.
5. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
6. The dsRNA of claim 1, wherein the GalNAc (N-acetylgalactosamine) derivative is attached through a bivalent or trivalent branched linker.
7. The dsRNA agent of claim 1, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of
(SEQ ID NO: 387)
5′-CAUAUUUGAUCAGUCUUUUUA-3′
and
(SEQ ID NO: 572)
5′-UAAAAAGACUGAUCAAAUAUGUU-3′;
(SEQ ID NO: 287)
5′-ACAUAUUUGAUCAGUCUUUUU-3′
and
(SEQ ID NO: 472)
5′-AAAAAGACUGAUCAAAUAUGUUG-3′;
(SEQ ID NO: 303)
5′-AACAUAUUUGAUCAGUCUUUU-3′
and
(SEQ ID NO: 488)
5′-AAAAGACUGAUCAAAUAUGUUGA-3′;
(SEQ ID NO: 39)
5′-ACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 101)
5′-AAAGACUGAUCAAAUAUGU-3′;
(SEQ ID NO: 294)
5′-CAACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 479)
5′-AAAGACUGAUCAAAUAUGUUGAG-3′;
(SEQ ID NO: 358)
5′-UCAACAUAUUUGAUCAGUCUU-3′
and
(SEQ ID NO: 543)
5′-AAGACUGAUCAAAUAUGUUGAGU-3′;
and
(SEQ ID NO: 64)
5′-CAACAUAUUUGAUCAGUCU-3′
and
(SEQ ID NO: 126)
5′-AGACUGAUCAAAUAUGUUG-3′.
8. A cell containing the dsRNA agent of claim 1.
9. A pharmaceutical composition for inhibiting expression of an ANGPTL3 gene, comprising the dsRNA agent of claim 1.
10. The pharmaceutical composition of claim 9, wherein the dsRNA agent is present in a buffered solution.
11. A method of inhibiting ANGPTL3 expression in a cell, the method comprising:
(a) contacting the cell with the dsRNA agent of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
12. The method of claim 11, wherein the cell is within a subject.
13. A method of inhibiting the expression of ANGPTL3 in a subject, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby inhibiting the expression of ANGPTL3 in the subject.
14. A method of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating the subject.
15. The method of claim 14, wherein the disorder is a disorder of lipid metabolism.
16. The method of claim 14, wherein the disorder is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, atherosclerosis, diabetes, cardiovascular disease, and coronary artery disease.
17. The method of claim 14, further comprising administering an additional therapeutic to the subject.
18. The method of claim 17, wherein the additional therapeutic is a statin.
19. The method of claim 14, wherein the dsRNA agent is administered at a dose of about 0.5 mg/kg to about 50 mg/kg.
20. The method of claim 14, wherein the administration of the dsRNA agent to the subject causes a decrease in one or more serum lipid and/or a decrease in ANGPTL3 protein accumulation.
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20
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RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 17/089,854, filed on Nov. 5, 2020, which is a continuation of U.S. patent application Ser. No. 16/411,261, filed on May 14, 2019, now, U.S. Pat. No. 10,934,545, issued on Mar. 2, 2021, which is a continuation of U.S. patent application Ser. No. 15/683,999, filed on Aug. 23, 2017, now U.S. Pat. No. 10,337,010, issued on Jul. 2, 2019, which is a continuation of U.S. patent application Ser. No. 15/068,912, now U.S. Pat. No. 9,771,591, issued on Sep. 26, 2017, which is a continuation of U.S. patent application Ser. No. 14/132,999 filed on Dec. 18, 2013, now U.S. Pat. No. 9,322,018, issued on Apr. 26, 2016, which is a 35 U.S.C. 111(a) continuation application, which claims priority to PCT/US2012/043378, filed on Jun. 20, 2012, U.S. Provisional Application No. 61/499,620, filed on Jun. 21, 2011, and to U.S. Provisional Application No. 61/638,288, filed on Apr. 25, 2012. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 19, 2021, is named 121301_00310_SL.txt and is 444,512 bytes in size.
BACKGROUND OF THE INVENTION
Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulates lipid metabolism and that is predominantly expressed in the liver (Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activities of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and of endothelial lipase (EL), which hydrolyzes high density lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese, KK/Snk mice, a reduction in ANGPTL3 expression has a protective effect against hyperlipidemia and artherosclerosis by promoting the clearance of triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223). Human ANGPTL3 plasma concentrations positively correlate with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).
Disorders of lipid metabolism can lead to elevated levels of serum lipids, such as triglycerides and/or cholesterol. Elevated serum lipids are strongly associated with high blood pressure, cardiovascular disease, diabetes and other pathologic conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder that is characterized by high blood levels of triglycerides. It has been associated with atherosclerosis, even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia can also lead to pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder that is characterized by elevated levels of any one or all lipids and/or lipoproteins in the blood. Current treatments for disorders of lipid metabolism, including dieting, exercise and treatment with statins and other drugs, are not always effective. Accordingly, there is a need in the art for alternative treatments for subjects having disorders of lipid metabolism.
SUMMARY OF THE INVENTION
The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPL3 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ANGPL3 gene, e.g., a subject suffering or prone to suffering from a disorder of lipid metabolism, such as a subject suffering or prone to suffering from hyperlipidemia or hypertriglyceridemia.
Accordingly, in one aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.
In another aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.
In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of AD-53063.1, AD-53001.1, AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 of Tables 7 and 8.
In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length.
In one embodiment, each strand of a dsRNA is no more than 30 nucleotides in length.
At least one strand of a dsRNA may comprise a 3′ overhang of at least 1 nucleotide or at least 2 nucleotides.
In certain embodiments, a dsRNA further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
In some embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is
In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic
In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.
In one embodiment, the region of complementarity of a dsRNA consists of one of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, a dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10.
In another aspect, the present invention provides a cell, e.g., a hepatocyte, containing a dsRNA of the invention.
In yet another aspect, the present invention provides a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage. The region of complementarity may be least 15 nucleotides in length or 19 to 21 nucleotides in length. In a further aspect, the present invention provides a cell comprising a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.
In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising a dsRNA or vector of the invention.
In one embodiment, the pharmaceutical composition comprises a lipid formulation, such as a MC3, SNALP or XTC formulation.
In another aspect, the present invention provides methods of inhibiting ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA or a vector of the invention, and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
The cell may be within a subject, such as a human subject, for example a human subject suffering from a disorder of lipid metabolism, e.g., hyperlipidemia or hypertriglyceridemia.
In one embodiment of the methods of the invention, ANGPTL3 expression is inhibited by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby treating the subject.
The disorder may be disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia
In one embodiment, the administration of the dsRNA to the subject causes a decrease in the level of a serum lipid, triglycerides, cholesterol and/or free fatty acids; and/or a decrease in ANGPTL3 protein accumulation. In one embodiment, administration of the dsRNA to the subject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, IDL-C and/or total cholesterol.
In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another aspect, the present invention provides methods of inhibiting the expression of ANGPTL3 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby inhibiting the expression of ANGPTL3 in the subject.
In yet another aspect, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method of inhibiting expression of ANGPTL3 gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the ANGPTL3 in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of the experimental procedure used for in vivo tests described in Example 2.
FIG. 2A is a graph showing measured levels of ANGPTL3 protein in WT mice after treatment with the indicated iRNA or a control.
FIG. 2B is a graph showing measured levels of ANGPTL3 protein in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 3A is a graph showing measured levels of LDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 3B is a graph showing measured levels of LDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 4A is a graph showing measured levels of triglycerides in WT mice after treatment with the indicated iRNA or a control.
FIG. 4B is a graph showing measured levels of triglycerides in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 5A is a graph showing measured levels of total cholesterol (TC) in WT mice after treatment with the indicated iRNA or a control.
FIG. 5B is a graph showing measured levels of total cholesterol (TC) in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 6A is a graph showing measured levels of HDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 6B is a graph showing measured levels of HDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 7 is a graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or a control.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPTL3gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ANGPTL3 gene. The use of these iRNAs enables the targeted degradation of mRNAs of an ANGPTL3 gene in mammals. Very low dosages of ANGPTL3 iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of an ANGPTL3 gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of expression of an ANGPTL3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of an ANGPTL3 protein, such as a subject having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ANGPTL3 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.
I. Definitions
In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
The term “ANGPTL3” refers to an angiopoietin like protein 3 having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3. The term encompasses full-length unprocessed precursor forms of ANGPTL3 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the fibrinogen-like domain. The sequence of a human ANGPTL3 mRNA transcript can be found at, for example, GenBank Accession No. GI: 41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesus ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 142388354 (NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 68163568 (NM_001025065.1; SEQ ID NO:4).
The term“ANGPTL3” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the ANGPTL3 gene, such as a single nucleotide polymorphism in the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the ANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039; rs192778191; rs192764027; rs192528948; rs191931953; rs191293319; rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3gene.
The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of ANGPTL3 in a cell, e.g., a cell within a subject, such as a mammalian subject.
In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an ANGPTL3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.
In another aspect, the RNAi agent is a single-stranded antisense RNA molecule. An antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule may be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an ANGPTL3 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.
As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an ANGPTL3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.
The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a part of an ANGPTL3mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ANGPTL3.
In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.
The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of an ANGPTL3,” as used herein, includes inhibition of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 gene that encode an ANGPTL3 protein.
“Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
The expression of an ANGPTL3 gene may be assessed based on the level of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of an ANGPTL3 may also be assessed indirectly based on the levels of a serum lipid, a triglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
In one embodiment, at least partial suppression of the expression of an ANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNA which can be isolated from or detected in a first cell or group of cells in which an ANGPTL3 gene is transcribed and which has or have been treated such that the expression of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:
(
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-
(
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(
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100
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The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.
In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.
The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817, the entire contents of which are hereby incorporated herein by reference. Examples of “SNALP” formulations are described below.
As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human having a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as lowering levels of triglycerides in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a disorder of lipid metabolism, such as, e.g., a decrease in the size of eruptive xanthomas. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., high triglyceride levels or eruptive xanthoma. The likelihood of developing a high tryglyceride levels or eruptive xanthoma is reduced, for example, when an individual having one or more risk factors for a high tryglyceride levels or eruptive xanthoma either fails to develop high tryglyceride levels or eruptive xanthoma or develops high tryglyceride levels or eruptive xanthoma with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition i (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.
As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e.g., a lipoprotein complex. Non-limiting examples of serum lipids may include triglycerides and cholesterol, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).
As used herein, a “disorder of lipid metabolism” refers to any disorder associated with or caused by a disturbance in lipid metabolism. For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, or an acquired disorder, such as a disorder acquired as a result of a diet or intake of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrom, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).
Cardiovascular diseases associated with disorders of lipid metabolism are also considered “disorders of lipid metabolism”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke.
Disorders related to body weight are also considered “disorders of lipid metabolism”, as defined herein. Such disorders may include obesity, metabolic syndrome including independent components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss.
Blood sugar disorders are further considered “disorders of lipid metabolism”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of lipid metabolism may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.
“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a disorder of lipid metabolism, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a disorder of lipid metabolism, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.
II. iRNAs of the Invention
Described herein are iRNAs which inhibit the expression of an ANGPTL3 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a disorder of lipid metabolism, e.g., familial hyperlipidemia. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ANGPTL3gene, The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, a primate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.
A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an ANGPTL3gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ANGPTL3 expression is not generated in the target cell by cleavage of a larger dsRNA.
A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.
In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in Tables 2, 3, 7, 8, 9 and 10, and the corresponding antisense strand of the sense strand is selected from the group of sequences of Tables 2, 3, 7, 8, 9 and 10. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an ANGPTL3gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in Tables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.
The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 7, 8, 9 and 10, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2, 3, 7, 8, 9 and 10 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 2, 3, 7, 8, 9 and 10, and differing in their ability to inhibit the expression of an ANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.
In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify a site(s) in an ANGPTL3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 7, 8, 9 and 10 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ANGPTL3gene.
While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and 10 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 7, 8, 9 and 10, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.
An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an ANGPTL3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ANGPTL3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ANGPTL3 gene is important, especially if the particular region of complementarity in an ANGPTL3 gene is known to have polymorphic sequence variation within the population.
III. Modified iRNAs of the Invention
In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2)−nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.
Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.
IV. iRNAs Conjugated to Ligands
Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.
Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
A. Lipid Conujugates
In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).
B. Cell Permeation Agents
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as t at or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
C. Carbohydrate Conjugates
In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as
In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:
Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,
(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.
In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
D. Linkers
In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.
The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
i. Redox Cleavable Linking Groups
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
ii. Phosphate-Based Cleavable Linking Groups
In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.
iii. Acid Cleavable Linking Groups
In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
iv. Ester-Based Linking Groups
In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
v. Peptide-Based Cleaving Groups
In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,
when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):
wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), CC or C(O); R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,
or heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):
wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II_VII, XI, X, and XIII
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.
“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
IV. Delivery of an iRNA of the Invention
The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
A. Vector Encoded iRNAs of the Invention
iRNA targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., (1993) Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood 83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; and Wang et al., (1995) Gene Therapy 2:775-783. A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
V. Pharmaceutical Compositions of the Invention
The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, such as hypertriglyceridemia.
Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.
The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a ANGPTL3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on ANGPTL3 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of lipid metabolism that would benefit from reduction in the expression of ANGPTL3. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of an LDL receptor (LDLR−/− mouse; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced artherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).
The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
A. iRNA Formulations Comprising Membranous Molecular Assemblies
An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.
A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.
Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).
Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.
Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.
Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
B. Nucleic Acid Lipid Particles
iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.
The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary lipid-dsRNA formulations are described in the table below.
cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid
conjugate
Ionizable/Cationic Lipid
Lipid:siRNA ratio
SNALP-1
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA
dimethylaminopropane (DLinDMA)
(57.1/7.1/34.4/1.4)
lipid:siRNA ~7:1
2-XTC
2,2-Dilinoleyl-4-
XTC/DPPC/Cholesterol/PEG-cDMA
dimethylaminoethyl-[1,3]-dioxolane
57.1/7.1/34.4/1.4
(XTC)
lipid:siRNA ~7:1
LNP05
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG
dimethylaminoethyl-[1,3]-dioxolane
57.5/7.5/31.5/3.5
(XTC)
lipid:siRNA ~6:1
LNP06
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG
dimethylaminoethyl-[1,3]-dioxolane
57.5/7.5/31.5/3.5
(XTC)
lipid:siRNA ~11:1
LNP07
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG
dimethylaminoethyl-[1,3]-dioxolane
60/7.5/31/1.5,
(XTC)
lipid:siRNA ~6:1
LNP08
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG
dimethylaminoethyl-[1,3]-dioxolane
60/7.5/31/1.5,
(XTC)
lipid:siRNA ~11:1
LNP09
2,2-Dilinoleyl-4-
XTC/DSPC/Cholesterol/PEG-DMG
dimethylaminoethyl-[1,3]-dioxolane
50/10/38.5/1.5
(XTC)
Lipid:siRNA 10:1
LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG
di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5
dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1
cyclopenta[d][1,3]dioxol-5-amine
(ALN100)
LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG
6,9,28,31-tetraen-19-yl
50/10/38.5/1.5
4-(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1
LNP12
1,1′-(2-(4-(2-((2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-DMG
hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5
hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1
1-yl)ethylazanediyl)didodecan-
2-ol (Tech G1)
LNP13
XTC
XTC/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 33:1
LNP14
MC3
MC3/DSPC/Chol/PEG-DMG
40/15/40/5
Lipid:siRNA: 11:1
LNP15
MC3
MC3/DSPC/Chol/PEG-
DSG/GalNAc-PEG-DSG
50/10/35/4.5/0.5
Lipid:siRNA: 11:1
LNP16
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP17
MC3
MC3/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP18
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 12:1
LNP19
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/35/5
Lipid:siRNA: 8:1
LNP20
MC3
MC3/DSPC/Chol/PEG-DPG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP22
XTC
XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
DSPC: distearoylphosphatidylcholine
DPPC: dipalmitoylphosphatidylcholine
PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.
ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
Synthesis of Ionizable/Cationic Lipids
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
Synthesis of 515
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).
Synthesis of 517A and 517B
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an .Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield:—6 g crude
517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS-[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR 6=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
C. Additional Formulations
i. Emulsions
The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
ii. Microemulsions
In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
iii. Microparticles
an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
iv. Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstras se, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.
Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
v. Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
vi. Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
vii. Other Components
The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a disorder of lipid metabolism. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by ANGPTL3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
VI. Methods of the Invention
The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression of ANGPTL3 may also be assessed indirectly by measuring a decrease in biological activity of ANGPTL3, e.g., a decrease in the level of serum lipid, triglycerides, cholesterol and/or free fatty acids.
In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
A cell suitable for treatment using the methods of the invention may be any cell that expresses an ANGPTL3gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.
ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.
The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ANGPTL3 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.
In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.
In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.
The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
In one aspect, the present invention also provides methods for inhibiting the expression of an ANGPTL3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an ANGPTL3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in ANGPTL3 gene and/or protein expression.
The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of ANGPTL3 expression, in a therapeutically effective amount of an iRNA targeting an ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting an ANGPTL3 gene.
An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.
Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
Subjects that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression are those having a disorder of lipid metabolism, e.g., an inherited disorder of lipid metabolism or an acquired disorder of lipid metabolism. In one embodiment, a subject having disorder of lipid metabolism has hyperlipidemia. In another embodiment, a subject having a disorder of lipid metabolism has hypertriglyceridemia. Treatment of a subject that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., a subject is having eruptive xanthomas) and prophylactic treatment (e.g., the subject is not having eruptive xanthomas or a subject may be at risk of developing eruptive xanthomas).
The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting ANGPTL3 is administered in combination with, e.g., an agent useful in treating a disorder of lipid metabolism as described elsewhere herein. For example, additional agents suitable for treating a subject that would benefit from reducton in ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, may include agents that lower one or more serum lipids. Non-limiting examples of such agents may include cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors, e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). Other agents useful in treating a disorder of lipid metabolism may include bile sequestering agents, such as cholestyramine and other resins; VLDL secretion inhibitors, such as niacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterol acyl transferase inhibitors; farnesoid X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptide; and therapeutic antibodies against ANGPTL3. The additional therapeutic agents may also include agents that raise high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, the additional therapeutic agents may also include dietary supplements, e.g., fish oil. The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.
In one embodiment, the method includes administering a composition featured herein such that expression of the target ANGPTL3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or abour 36 hours. In one embodiment, expression of the target ANGPTL3 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.
Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target ANGPTL3gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.
Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ANGPTL3 or pharmaceutical composition thereof, “effective against” a disorder of lipid metabolism indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating disorder of lipid metabolisms and the related causes.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.
Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of dsRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of dsRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.
Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 days. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some embodiments, the dosing may comprise a loading phase of multiple doses on consequitive days.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Transcripts
siRNA design was carried out to identify siRNAs targeting the human ANGPTL3 transcript annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/) and a cynomolgus monkey (Macaca fascicularis; henceforth “cyno”) ANGPTL3 transcript produced via sequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was done in-house, and the mRNA sequence is shown in SEQ ID NO:9. Design used the following transcripts from the NCBI collection: Human—NM_014495.2 (SEQ ID NO:1); Mouse—NM_013913.3 (SEQ ID NO:2). All siRNA duplexes were designed that shared 100% identity with the listed human and cyno transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in NCBI Gene database.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 977 candidate human/cyno siRNAs, and a subset of 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) were then used in a comprehensive search against the human transcriptome (defined as the set of NM_ and XM_records within the human NCBI Refseq set) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start were used to create 2 heptamers and one octomer. ‘Heptamer1’ was created by adding a 3′ A to the hexamer; ‘heptamer2’ was created by adding a 5′ A to the hexamer; octomer was created by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3×normalized octomer count)+(2×heptamer2 count)+(1×heptamer1 count)).
Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. Sorting was carried out by the specificity of the antisense strand. Duplexes were then selected from the human/cyno set with antisense oligos lacking miRNA seed matches, scores of 3 or better, less than 65% overall GC content, no GC at the first position, 4 or more Us or As in the seed region, and GC at the nineteenth position. Duplexes from the human/cyno/mouse set with antisense oligos having scores of 2 or better, less than 65% overall GC content, and no GC at the first position were also selected.
siRNA Sequence Selection
A total of 47 sense and 47 antisense derived siRNA oligos from the human/cyno set were synthesized and formed into duplexes. A total of 15 sense and 15 antisense derived siRNAs from the human/cyno/mouse set were synthesized and formed into duplexes.
Synthesis of ANGPTL3 Sequences
ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either a 1 or 0.2 μmol scale. Single strands were synthesized with 2′O-methyl modifications for transfection based in vitro screening. For use in free uptake screening assays, 3′ GalNAc conjugates were made with 2′F and 2′-O-methyl chemical modifications. In these designs, GalNAc moiety was placed at the 3′end of the sense strand. The antisense sequence was 23 nucleotides in length and also contained 2′F and 2′Omethyl chemical modifications with two phosphorothioate linkages at the 3′end.
On one set of 21mer single strands and duplexes, ‘endolight’ chemistry was applied as detailed below.
All pyrimidines (cytosine and uridine) in the sense strand were modified with 2′-O-Methyl nucleotides (2′ 0-Methyl C and 2′-O-Methyl U)
In the antisense strand, pyrimidines adjacent (towards 5′ position) to ribo A nucleoside were replaced with their corresponding 2′-O-Methyl nucleosides
A two base dTsdT extension at the 3′ end of both sense and anti sense sequences was introduced
For GalNAc conjugated 21mer sense and complementary 23mer antisense sequences, 2′F and 2′OMethyl modified single strands were synthesized. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support for the antisense sequence at a 1 μmol scale. The sequence motif named TOFFEE was applied, in which the sense strand contained a three-nucleotide 2′F-modified motif at positions 9, 10 and 11 and in the antisense, a 2′OMethyl-modified motif was included at positions 11, 12 and 13.
Synthesis, Cleavage and Deprotection
The synthesis of ANGPTL3 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for the sense strand and a universal CPG for the antisesense strand were used.
The synthesis of the above sequences was performed at either a 1 or 0.2 μm scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as the activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mix and the pellets were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification, Desalting and Annealing
ANGPTL3 sequences were precipitated and purified on an AKTA Purifier system using a Sephadex column. The ANGPTL3 was run at ambient temperature. Sample injection and collection was performed in 96 well plates with 1.8 mL deep wells. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ANGPTL3 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.
Example 2. In Vitro Screening
Cell Culture and Transfections
Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×104 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.
Free Uptake Transfection
5 μl of each GalNac conjugated siRNA in PBS was combined with 4×104 freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspended in 95 μl of In Vitro Gro CP media (In Vitro Technologies-Celsis, Baltimore, Md.) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37° C. in an atmosphere of 5% CO2. siRNAs were tested at final concentrations of 500 nM, 100 nM and 10 nM for efficacy free uptake assays. For dose response screens, final siRNA concentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl of Wash Buffer B, captured, and the supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured, and the supernatant was removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR
2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat #Hs00205581_m1) and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).
Viability Screens
Cell viability was measured on days 3 and 6 in HeLa and Hep3B cells following transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells were plated at a density of 10,000 cells per well in 96 well plates. Each siRNA was assayed in triplicate and the data averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability, and AD-1955 and mock transfected cells as negative controls. PLK1 and AD-19200 result in a dose dependent loss of viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96 well plates after 3 or 6 days and incubated at 37° C. for 2 hours. Plates were then read in a Spectrophotometer (Molecular Devices) at 560Ex/590Em. Viability was expressed as the average value of light units from three replicate transfections+/−standard deviation. Relative viability was assessed by first averaging the three replicate transfections and then normalizing Mock transfected cells. Data is expressed as % viabile cells.
TABLE 1
Abbreviations of nucleotide monomers used
in nucleic acid sequence representation.
It will be understood that these monomers, when
present in an oligonucleotide, are mutually
linked by 5′-3′-phosphodiester bonds.
Abbreviation
Nucleotide(s)
A
adenosine
C
cytidine
G
guanosine
T
thymidine
U
uridine
N
any nucleotide (G, A, C, T or U)
a
2′-O-methyladenosine
c
2′-O-methylcytidine
g
2′-O-methylguanosine
u
2′-O-methyluridine
dT
2′-deoxythymidine
s
phosphorothioate linkage
TABLE 2
Unmodified sense and antisense strand sequences of ANGPTL3 dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 16-77,
(SEQ ID NOS 78-139,
Sense
respectively,
Position in
Antisense
respectively,
Position in
Duplex ID
Name
in order of appearance)
NM_014495.2 _
Name
in order of appearance)
NM_014495.2
AD-45939.1
A-96225.1
UAUUUGAUCAGUCUUUUUA
281-299
A-96226.1
UAAAAAGACUGAUCAAAUA
281-299
AD-45858.1
A-96149.1
GAGCAACUAACUAACUUAA
478-496
A-96150.1
UUAAGUUAGUUAGUUGCUC
478-496
AD-45869.1
A-96137.1
GGCCAAAUUAAUGACAUAU
247-265
A-96138.1
AUAUGUCAUUAAUUUGGCC
247-265
AD-45884.1
A-96189.1
CGAAUUGAGUUGGAAGACU
1045-1063
A-96190.1
AGUCUUCCAACUCAAUUCG
1045-1063
AD-45892.1
A-96129.1
CCUCCUUCAGUUGGGACAU
198-216
A-96130.1
AUGUCCCAACUGAAGGAGG
198-216
AD-45899.1
A-96147.1
CACUUGAACUCAACUCAAA
401-419
A-96148.1
UUUGAGUUGAGUUCAAGUG
401-419
AD-45915.1
A-96231.1
GUCCAUGGACAUUAAUUCA
890-908
A-96232.1
UGAAUUAAUGUCCAUGGAC
890-908
AD-45924.1
A-96219.1
AAUCAAGAUUUGCUAUGUU
152-170
A-96220.1
AACAUAGCAAAUCUUGAUU
152-170
AD-45860.1
A-96181.1
CUAGAGAAGAUAUACUCCA
1000-1018
A-96182.1
UGGAGUAUAUCUUCUCUAG
1000-1018
AD-45870.1
A-96153.1
CUAACUAACUUAAUUCAAA
484-502
A-96154.1
UUUGAAUUAAGUUAGUUAG
484-502
AD-45870.2
A-96153.2
CUAACUAACUUAAUUCAAA
484-502
A-96154.2
UUUGAAUUAAGUUAGUUAG
484-502
AD-45877.1
A-96171.1
CAUUAAUUCAACAUCGAAU
899-917
A-96172.1
AUUCGAUGUUGAAUUAAUG
899-917
AD-45885.1
A-96205.1
CAAAAUGUUGAUCCAUCCA
1392-1410
A-96206.1
UGGAUGGAUCAACAUUUUG
1392-1410
AD-45893.1
A-96145.1
CAUAUAAACUACAAGUCAA
359-377
A-96146.1
UUGACUUGUAGUUUAUAUG
359-377
AD-45900.1
A-96163.1
GACCCAGCAACUCUCAAGU
839-857
A-96164.1
ACUUGAGAGUUGCUGGGUC
839-857
AD-45925.1
A-96235.1
GGUUGGGCCUAGAGAAGAU
992-1010
A-96236.1
AUCUUCUCUAGGCCCAACC
992-1010
AD-45861.1
A-96197.1
GUGUGGAGAAAACAACCUA
1272-1290
A-96198.1
UAGGUUGUUUUCUCCACAC
1272-1290
AD-45871.1
A-96169.1
GACAUUAAUUCAACAUCGA
897-915
A-96170.1
UCGAUGUUGAAUUAAUGUC
897-915
AD-45878.1
A-96187.1
CAUAGUGAAGCAAUCUAAU
1017-1035
A-96188.1
AUUAGAUUGCUUCACUAUG
1017-1035
AD-45886.1
A-96127.1
CUAUGUUAGACGAUGUAAA
164-182
A-96128.1
UUUACAUCGUCUAACAUAG
164-182
AD-45894.1
A-96161.1
CACAGAAAUUUCUCUAUCU
684-702
A-96162.1
AGAUAGAGAAAUUUCUGUG
684-702
AD-45901.1
A-96179.1
GUUGGGCCUAGAGAAGAUA
993-1011
A-96180.1
UAUCUUCUCUAGGCCCAAC
993-1011
AD-45909.1
A-96213.1
GCCAAAAUCAAGAUUUGCU
147-165
A-96214.1
AGCAAAUCUUGAUUUUGGC
147-165
AD-45934.1
A-96223.1
ACAUAUUUGAUCAGUCUUU
278-296
A-96224.1
AAAGACUGAUCAAAUAUGU
278-296
AD-45934.2
A-96223.2
ACAUAUUUGAUCAGUCUUU
278-296
A-96224.2
AAAGACUGAUCAAAUAUGU
278-296
AD-45863.1
A-96135.1
CUUAAAGACUUUGUCCAUA
220-238
A-96136.1
UAUGGACAAAGUCUUUAAG
220-238
AD-45872.1
A-96185.1
CCAUAGUGAAGCAAUCUAA
1016-1034
A-96186.1
UUAGAUUGCUUCACUAUGG
1016-1034
AD-45879.1
A-96203.1
CAACCAAAAUGUUGAUCCA
1388-1406
A-96204.1
UGGAUCAACAUUUUGGUUG
1388-1406
AD-45887.1
A-96143.1
CUACAUAUAAACUACAAGU
356-374
A-96144.1
ACUUGUAGUUUAUAUGUAG
356-374
AD-45895.1
A-96177.1
GGGAGGCUUGAUGGAGAAU
970-988
A-96178.1
AUUCUCCAUCAAGCCUCCC
970-988
AD-45902.1
A-96195.1
GGUGUUUUCUACUUGGGAU
1188-1206
A-96196.1
AUCCCAAGUAGAAAACACC
1188-1206
AD-45910.1
A-96229.1
AAGAGCACCAAGAACUACU
711-729
A-96230.1
AGUAGUUCUUGGUGCUCUU
711-729
AD-45935.1
A-96239.1
UGGAGAAAACAACCUAAAU
1275-1293
A-96240.1
AUUUAGGUUGUUUUCUCCA
1275-1293
AD-45864.1
A-96151.1
GCAACUAACUAACUUAAUU
480-498
A-96152.1
AAUUAAGUUAGUUAGUUGC
480-498
AD-45873.1
A-96201.1
CAACCUAAAUGGUAAAUAU
1284-1302
A-96202.1
AUAUUUACCAUUUAGGUUG
1284-1302
AD-45880.1
A-96125.1
GCUAUGUUAGACGAUGUAA
163-181
A-96126.1
UUACAUCGUCUAACAUAGC
163-181
AD-45888.1
A-96159.1
CCCACAGAAAUUUCUCUAU
682-700
A-96160.1
AUAGAGAAAUUUCUGUGGG
682-700
AD-45896.1
A-96193.1
GAUUUGGUGUUUUCUACUU
1183-1201
A-96194.1
AAGUAGAAAACACCAAAUC
1183-1201
AD-45903.1
A-96211.1
CAGAGCCAAAAUCAAGAUU
143-161
A-96212.1
AAUCUUGAUUUUGGCUCUG
143-161
AD-45919.1
A-96217.1
AAAUCAAGAUUUGCUAUGU
151-169
A-96218.1
ACAUAGCAAAUCUUGAUUU
151-169
AD-45865.1
A-96167.1
CAUGGACAUUAAUUCAACA
893-911
A-96168.1
UGUUGAAUUAAUGUCCAUG
893-911
AD-45874.1
A-96123.1
GAUUUGCUAUGUUAGACGA
158-176
A-96124.1
UCGUCUAACAUAGCAAAUC
158-176
AD-45881.1
A-96141.1
GAACUACAUAUAAACUACA
353-371
A-96142.1
UGUAGUUUAUAUGUAGUUC
353-371
AD-45889.1
A-96175.1
CGAAUAGAUGGAUCACAAA
913-931
A-96176.1
UUUGUGAUCCAUCUAUUCG
913-931
AD-45897.1
A-96209.1
CUUGUUAAAACUCUAAACU
1817-1835
A-96210.1
AGUUUAGAGUUUUAACAAG
1817-1835
AD-45904.1
A-96227.1
AUUUGAUCAGUCUUUUUAU
282-300
A-96228.1
AUAAAAAGACUGAUCAAAU
282-300
AD-45920.1
A-96233.1
UCCAUGGACAUUAAUUCAA
891-909
A-96234.1
UUGAAUUAAUGUCCAUGGA
891-909
AD-45856.1
A-96117.1
CACAAUUAAGCUCCUUCUU
57-75
A-96118.1
AAGAAGGAGCUUAAUUGUG
57-75
AD-45929.1
A-96221.1
CAACAUAUUUGAUCAGUCU
276-294
A-96222.1
AGACUGAUCAAAUAUGUUG
276-294
AD-45866.1
A-96183.1
CUCCAUAGUGAAGCAAUCU
1014-1032
A-96184.1
AGAUUGCUUCACUAUGGAG
1014-1032
AD-45875.1
A-96139.1
GCCAAAUUAAUGACAUAUU
248-266
A-96140.1
AAUAUGUCAUUAAUUUGGC
248-266
AD-45882.1
A-96157.1
CAACAGCAUAGUCAAAUAA
622-640
A-96158.1
UUAUUUGACUAUGCUGUUG
622-640
AD-45890.1
A-96191.1
GGAAAUCACGAAACCAACU
1105-1123
A-96192.1
AGUUGGUUUCGUGAUUUCC
1105-1123
AD-45898.1
A-96131.1
CAGUUGGGACAUGGUCUUA
205-223
A-96132.1
UAAGACCAUGUCCCAACUG
205-223
AD-45857.1
A-96133.1
GACAUGGUCUUAAAGACUU
212-230
A-96134.1
AAGUCUUUAAGACCAUGUC
212-230
AD-45930.1
A-96237.1
UGUGGAGAAAACAACCUAA
1273-1291
A-96238.1
UUAGGUUGUUUUCUCCACA
1273-1291
AD-45867.1
A-96199.1
GUGGAGAAAACAACCUAAA
1274-1292
A-96200.1
UUUAGGUUGUUUUCUCCAC
1274-1292
AD-45876.1
A-96155.1
CCAACAGCAUAGUCAAAUA
621-639
A-96156.1
UAUUUGACUAUGCUGUUGG
621-639
AD-45883.1
A-96173.1
CAACAUCGAAUAGAUGGAU
907-925
A-96174.1
AUCCAUCUAUUCGAUGUUG
907-925
AD-45891.1
A-96207.1
GCAAAUUUAAAAGGCAAUA
1441-1459
A-96208.1
UAUUGCCUUUUAAAUUUGC
1441-1459
AD-45914.1
A-96215.1
CAAAAUCAAGAUUUGCUAU
149-167
A-96216.1
AUAGCAAAUCUUGAUUUUG
149-167
AD-15838.1
A-26242.1
AGAGCCAAAAUCAAGAUUU
144-162
A-26243.2
AAAUCUUGAUUUUGGCUCU
144-162
TABLE 3
Modified sense and antisense strand sequences of ANGPTL3 dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 140-201,
(SEQ ID NOS 202-263,
respectively,
respectively,
Duplex ID
Sense OligoName
in order of appearance)
Antisense OligoName
in order of appearance)
AD-45939.1
A-96225.1
uAuuuGAucAGucuuuuuAdTsdT
A-96226.1
uAAAAAGACUGAUcAAAuAdTsdT
AD-45858.1
A-96149.1
GAGcAAcuAAcuAAcuuAAdTsdT
A-96150.1
UuAAGUuAGUuAGUUGCUCdTsdT
AD-45869.1
A-96137.1
GGccAAAuuAAuGAcAuAudTsdT
A-96138.1
AuAUGUcAUuAAUUUGGCCdTsdT
AD-45884.1
A-96189.1
cGAAuuGAGuuGGAAGAcudTsdT
A-96190.1
AGUCUUCcAACUcAAUUCGdTsdT
AD-45892.1
A-96129.1
ccuccuucAGuuGGGAcAudTsdT
A-96130.1
AUGUCCcAACUGAAGGAGGdTsdT
AD-45899.1
A-96147.1
cAcuuGAAcucAAcucAAAdTsdT
A-96148.1
UUUGAGUUGAGUUcAAGUGdTsdT
AD-45915.1
A-96231.1
GuccAuGGAcAuuAAuucAdTsdT
A-96232.1
UGAAUuAAUGUCcAUGGACdTsdT
AD-45924.1
A-96219.1
AAucAAGAuuuGcuAuGuudTsdT
A-96220.1
AAcAuAGcAAAUCUUGAUUdTsdT
AD-45860.1
A-96181.1
cuAGAGAAGAuAuAcuccAdTsdT
A-96182.1
UGGAGuAuAUCUUCUCuAGdTsdT
AD-45870.1
A-96153.1
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.1
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45870.2
A-96153.2
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.2
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45877.1
A-96171.1
cAuuAAuucAAcAucGAAudTsdT
A-96172.1
AUUCGAUGUUGAAUuAAUGdTsdT
AD-45885.1
A-96205.1
cAAAAuGuuGAuccAuccAdTsdT
A-96206.1
UGGAUGGAUcAAcAUUUUGdTsdT
AD-45893.1
A-96145.1
cAuAuAAAcuAcAAGucAAdTsdT
A-96146.1
UUGACUUGuAGUUuAuAUGdTsdT
AD-45900.1
A-96163.1
GAcccAGcAAcucucAAGudTsdT
A-96164.1
ACUUGAGAGUUGCUGGGUCdTsdT
AD-45925.1
A-96235.1
GGuuGGGccuAGAGAAGAudTsdT
A-96236.1
AUCUUCUCuAGGCCcAACCdTsdT
AD-45861.1
A-96197.1
GuGuGGAGAAAAcAAccuAdTsdT
A-96198.1
uAGGUUGUUUUCUCcAcACdTsdT
AD-45871.1
A-96169.1
GAcAuuAAuucAAcAucGAdTsdT
A-96170.1
UCGAUGUUGAAUuAAUGUCdTsdT
AD-45878.1
A-96187.1
cAuAGuGAAGcAAucuAAudTsdT
A-96188.1
AUuAGAUUGCUUcACuAUGdTsdT
AD-45886.1
A-96127.1
cuAuGuuAGAcGAuGuAAAdTsdT
A-96128.1
UUuAcAUCGUCuAAcAuAGdTsdT
AD-45894.1
A-96161.1
cAcAGAAAuuucucuAucudTsdT
A-96162.1
AGAuAGAGAAAUUUCUGUGdTsdT
AD-45901.1
A-96179.1
GuuGGGccuAGAGAAGAuAdTsdT
A-96180.1
uAUCUUCUCuAGGCCcAACdTsdT
AD-45909.1
A-96213.1
GccAAAAucAAGAuuuGcudTsdT
A-96214.1
AGcAAAUCUUGAUUUUGGCdTsdT
AD-45934.1
A-96223.1
AcAuAuuuGAucAGucuuudTsdT
A-96224.1
AAAGACUGAUcAAAuAUGUdTsdT
AD-45934.2
A-96223.2
AcAuAuuuGAucAGucuuudTsdT
A-96224.2
AAAGACUGAUcAAAuAUGUdTsdT
AD-45863.1
A-96135.1
cuuAAAGAcuuuGuccAuAdTsdT
A-96136.1
uAUGGAcAAAGUCUUuAAGdTsdT
AD-45872.1
A-96185.1
ccAuAGuGAAGcAAucuAAdTsdT
A-96186.1
UuAGAUUGCUUcACuAUGGdTsdT
AD-45879.1
A-96203.1
cAAccAAAAuGuuGAuccAdTsdT
A-96204.1
UGGAUcAAcAUUUUGGUUGdTsdT
AD-45887.1
A-96143.1
cuAcAuAuAAAcuAcAAGudTsdT
A-96144.1
ACUUGuAGUUuAuAUGuAGdTsdT
AD-45895.1
A-96177.1
GGGAGGcuuGAuGGAGAAudTsdT
A-96178.1
AUUCUCcAUcAAGCCUCCCdTsdT
AD-45902.1
A-96195.1
GGuGuuuucuAcuuGGGAudTsdT
A-96196.1
AUCCcAAGuAGAAAAcACCdTsdT
AD-45910.1
A-96229.1
AAGAGcAccAAGAAcuAcudTsdT
A-96230.1
AGuAGUUCUUGGUGCUCUUdTsdT
AD-45935.1
A-96239.1
uGGAGAAAAcAAccuAAAudTsdT
A-96240.1
AUUuAGGUUGUUUUCUCcAdTsdT
AD-45864.1
A-96151.1
GcAAcuAAcuAAcuuAAuudTsdT
A-96152.1
AAUuAAGUuAGUuAGUUGCdTsdT
AD-45873.1
A-96201.1
cAAccuAAAuGGuAAAuAudTsdT
A-96202.1
AuAUUuACcAUUuAGGUUGdTsdT
AD-45880.1
A-96125.1
GcuAuGuuAGAcGAuGuAAdTsdT
A-96126.1
UuAcAUCGUCuAAcAuAGCdTsdT
AD-45888.1
A-96159.1
cccAcAGAAAuuucucuAudTsdT
A-96160.1
AuAGAGAAAUUUCUGUGGGdTsdT
AD-45896.1
A-96193.1
GAuuuGGuGuuuucuAcuudTsdT
A-96194.1
AAGuAGAAAAcACcAAAUCdTsdT
AD-45903.1
A-96211.1
cAGAGccAAAAucAAGAuudTsdT
A-96212.1
AAUCUUGAUUUUGGCUCUGdTsdT
AD-45919.1
A-96217.1
AAAucAAGAuuuGcuAuGudTsdT
A-96218.1
AcAuAGcAAAUCUUGAUUUdTsdT
AD-45865.1
A-96167.1
cAuGGAcAuuAAuucAAcAdTsdT
A-96168.1
UGUUGAAUuAAUGUCcAUGdTsdT
AD-45874.1
A-96123.1
GAuuuGcuAuGuuAGAcGAdTsdT
A-96124.1
UCGUCuAAcAuAGcAAAUCdTsdT
AD-45881.1
A-96141.1
GAAcuAcAuAuAAAcuAcAdTsdT
A-96142.1
UGuAGUUuAuAUGuAGUUCdTsdT
AD-45889.1
A-96175.1
cGAAuAGAuGGAucAcAAAdTsdT
A-96176.1
UUUGUGAUCcAUCuAUUCGdTsdT
AD-45897.1
A-96209.1
cuuGuuAAAAcucuAAAcudTsdT
A-96210.1
AGUUuAGAGUUUuAAcAAGdTsdT
AD-45904.1
A-96227.1
AuuuGAucAGucuuuuuAudTsdT
A-96228.1
AuAAAAAGACUGAUcAAAUdTsdT
AD-45920.1
A-96233.1
uccAuGGAcAuuAAuucAAdTsdT
A-96234.1
UUGAAUuAAUGUCcAUGGAdTsdT
AD-45856.1
A-96117.1
cAcAAuuAAGcuccuucuudTsdT
A-96118.1
AAGAAGGAGCUuAAUUGUGdTsdT
AD-45929.1
A-96221.1
cAAcAuAuuuGAucAGucudTsdT
A-96222.1
AGACUGAUcAAAuAUGUUGdTsdT
AD-45866.1
A-96183.1
cuccAuAGuGAAGcAAucudTsdT
A-96184.1
AGAUUGCUUcACuAUGGAGdTsdT
AD-45875.1
A-96139.1
GccAAAuuAAuGAcAuAuudTsdT
A-96140.1
AAuAUGUcAUuAAUUUGGCdTsdT
AD-45882.1
A-96157.1
cAAcAGcAuAGucAAAuAAdTsdT
A-96158.1
UuAUUUGACuAUGCUGUUGdTsdT
AD-45890.1
A-96191.1
GGAAAucAcGAAAccAAcudTsdT
A-96192.1
AGUUGGUUUCGUGAUUUCCdTsdT
AD-45898.1
A-96131.1
cAGuuGGGAcAuGGucuuAdTsdT
A-96132.1
uAAGACcAUGUCCcAACUGdTsdT
AD-45857.1
A-96133.1
GAcAuGGucuuAAAGAcuudTsdT
A-96134.1
AAGUCUUuAAGACcAUGUCdTsdT
AD-45930.1
A-96237.1
uGuGGAGAAAAcAAccuAAdTsdT
A-96238.1
UuAGGUUGUUUUCUCcAcAdTsdT
AD-45867.1
A-96199.1
GuGGAGAAAAcAAccuAAAdTsdT
A-96200.1
UUuAGGUUGUUUUCUCcACdTsdT
AD-45876.1
A-96155.1
ccAAcAGcAuAGucAAAuAdTsdT
A-96156.1
uAUUUGACuAUGCUGUUGGdTsdT
AD-45883.1
A-96173.1
cAAcAucGAAuAGAuGGAudTsdT
A-96174.1
AUCcAUCuAUUCGAUGUUGdTsdT
AD-45891.1
A-96207.1
GcAAAuuuAAAAGGcAAuAdTsdT
A-96208.1
uAUUGCCUUUuAAAUUUGCdTsdT
AD-45914.1
A-96215.1
cAAAAucAAGAuuuGcuAudTsdT
A-96216.1
AuAGcAAAUCUUGAUUUUGdTsdT
AD-15838.1
A-26242.1
AGAGccAAAAucAAGAuuudTsdT
A-26243.2
AAAUCUuGAUUUuGGCUCUdTsdT
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides;
s is a phosphothiorate linkage.
TABLE 4
Results of single dose screen using ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide
duplexes listed in Table 3. The sequence of AD-15838.2 is
identical to the sequence of AD-15838.1. Delivery of siRNA
duplexes was done using LNPs.
Human Hep3B
Duplex
10 nM
0.1 nM
STDEV, 10 nM
STDEV, 0.1 nM
AD-15838.2
0.09
0.66
0.008
0.030
AD-45856.1
0.32
0.91
0.026
0.032
AD-45857.1
2.46
1.07
0.140
0.044
AD-45858.1
0.10
0.74
0.010
0.070
AD-45860.1
0.02
0.47
0.002
0.097
AD-45861.1
0.03
0.68
0.004
0.062
AD-45863.1
1.42
0.95
0.145
0.126
AD-45864.1
0.02
0.17
0.002
0.045
AD-45865.1
0.32
0.93
0.022
0.062
AD-45866.1
0.10
0.92
0.010
0.041
AD-45867.1
0.04
0.61
0.000
0.048
AD-45869.1
0.45
1.08
0.028
0.081
AD-45870.1
0.01
0.10
0.003
0.010
AD-45871.1
0.05
0.57
0.006
0.071
AD-45872.1
0.07
0.71
0.007
0.034
AD-45873.1
0.02
0.23
0.001
0.011
AD-45874.1
0.08
0.75
0.013
0.049
AD-45875.1
0.13
0.82
0.017
0.040
AD-45876.1
0.03
0.54
0.000
0.013
AD-45877.1
0.06
0.47
0.002
0.025
AD-45878.1
0.02
0.44
0.002
0.031
AD-45879.1
0.03
0.35
0.003
0.023
AD-45880.1
0.49
1.00
0.039
0.088
AD-45881.1
0.20
0.90
0.019
0.095
AD-45882.1
0.20
0.95
0.012
0.086
AD-45883.1
0.16
0.98
0.011
0.058
AD-45884.1
0.09
0.94
0.003
0.044
AD-45885.1
0.22
0.91
0.020
0.145
AD-45886.1
0.04
0.40
0.008
0.080
AD-45887.1
0.03
0.35
0.002
0.057
AD-45888.1
0.05
0.80
0.006
0.042
AD-45889.1
0.31
0.91
0.013
0.052
AD-45890.1
0.06
0.90
0.001
0.047
AD-45891.1
0.06
0.82
0.007
0.034
AD-45892.1
1.01
1.09
0.033
0.211
AD-45893.1
0.04
0.58
0.002
0.046
AD-45894.1
0.04
0.59
0.003
0.024
AD-45895.1
0.84
1.00
0.047
0.047
AD-45896.1
0.84
0.98
0.032
0.095
AD-45897.1
0.36
0.61
0.032
0.053
AD-45898.1
0.98
1.09
0.021
0.117
AD-45899.1
0.04
0.59
0.005
0.095
AD-45900.1
0.06
0.80
0.005
0.091
AD-45901.1
0.33
0.94
0.025
0.096
AD-45902.1
0.24
1.03
0.010
0.079
AD-45903.1
0.74
1.02
0.003
0.092
AD-45904.1
0.39
0.87
0.010
0.010
AD-45909.1
0.04
0.73
0.008
0.013
AD-45910.1
1.08
1.01
0.037
0.089
AD-45914.1
0.52
0.99
0.018
0.071
AD-45915.1
0.06
0.48
0.004
0.046
AD-45919.1
0.67
0.98
0.048
0.064
AD-45920.1
0.61
1.00
0.031
0.038
AD-45924.1
0.09
0.67
0.005
0.012
AD-45925.1
0.13
0.90
0.008
0.100
AD-45929.1
0.02
0.42
0.001
0.083
AD-45930.1
0.05
0.63
0.005
0.052
AD-45934.1
0.04
0.41
0.001
0.062
AD-45935.1
0.08
0.76
0.006
0.058
AD-45939.1
0.23
0.82
0.030
0.028
AD-1955.1
0.93
0.93
0.068
0.073
AD-1955.1
0.94
1.01
0.028
0.113
AD-1955.1
1.00
1.02
0.032
0.065
AD-1955.1
1.15
1.06
0.053
0.019
TABLE 5
Dose response screen results for ANGPTL3 dsRNA sequences
The experiments were conducted using modified
oligonucleotide duplexes listed in Table 3. The sequence
of AD-15838.2 is identical to the sequence of AD-15838.1.
Hep3B IC50
24 hrs
120 hrs
IC50
IC50
IC50 I
IC50 II
weighted
IC50 I
IC50 II
weighted
Duplex
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
AD-15838.2
0.027
0.006
0.017
0.657
0.937
0.800
AD-45860.1
0.006
0.002
0.004
0.045
0.032
0.039
AD-45864.1
0.002
0.001
0.002
0.046
0.042
0.044
AD-45870.1
0.002
0.001
0.001
0.011
0.008
0.010
AD-45873.1
0.005
0.004
0.005
0.037
0.025
0.031
AD-45876.1
0.032
0.006
0.019
0.269
0.045
0.156
AD-45877.1
0.018
0.012
0.015
1.660
0.538
1.091
AD-45878.1
0.023
0.015
0.019
0.252
0.131
0.190
AD-45879.1
0.002
0.003
0.003
0.023
0.029
0.026
AD-45886.1
0.004
0.004
0.004
0.030
0.018
0.025
AD-45887.1
0.010
0.009
0.010
0.058
0.059
0.059
AD-45915.1
0.016
0.015
0.015
0.110
0.056
0.083
AD-45929.1
0.023
0.008
0.016
0.227
0.025
0.124
AD-45934.1
0.006
0.006
0.006
0.110
0.045
0.077
TABLE 6
Results of cell viability screens using modified ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide duplexes listed
in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD-
15838.1. Viability data is expressed as % viable relative to mock treated cells.
Ave
Ave
Ave
Ave
Ave
SD
SD
SD
SD
SD
Target
Duplex
10 nM
1 nM
500 pM
100 pM
50 pM
10 nM
1 nM
500 pM
100 pM
50 pM
HeLa day 3
ANGPTL3
AD-15838.2
37.34
58.67
70.92
89.86
94.98
9.45
12.28
15.06
22.37
18.23
ANGPTL3
AD-15838.2
29.13
48.99
63.18
79.21
94.47
1.62
5.56
4.34
11.15
11.31
ANGPTL3
AD-45860.1
67.10
75.49
77.93
86.57
90.51
6.99
12.93
6.39
6.97
3.57
ANGPTL3
AD-45864.1
99.13
96.95
86.77
89.20
84.36
7.90
7.22
12.60
4.85
6.87
ANGPTL3
AD-45870.1
82.36
97.02
95.33
95.67
92.27
8.07
5.12
7.97
7.05
10.29
ANGPTL3
AD-45873.1
67.96
90.01
90.60
94.20
103.63
11.26
22.61
15.92
22.92
16.97
ANGPTL3
AD-45876.1
64.00
76.71
80.21
81.71
91.23
6.60
13.94
10.15
10.81
13.89
ANGPTL3
AD-45877.1
79.55
77.33
79.98
91.96
93.46
1.66
9.80
8.73
16.63
11.41
ANGPTL3
AD-45878.1
81.95
78.22
78.74
87.93
85.03
15.37
22.72
22.59
30.84
40.04
ANGPTL3
AD-45878.1
66.83
70.71
82.14
82.80
83.14
17.48
6.49
6.86
19.92
21.15
ANGPTL3
AD-45879.1
37.56
45.55
59.28
76.35
78.38
3.50
7.96
19.73
34.33
33.99
ANGPTL3
AD-45886.1
72.75
57.90
64.51
81.92
82.89
14.73
12.64
11.78
25.60
23.14
ANGPTL3
AD-45887.1
38.01
53.91
59.31
76.44
85.73
0.58
10.81
6.27
11.12
10.92
ANGPTL3
AD-45915.1
48.06
52.17
67.90
95.45
100.77
8.13
15.15
29.11
32.49
38.79
ANGPTL3
AD-45929.1
29.27
44.58
52.87
76.45
88.03
4.17
9.67
14.49
31.74
28.82
ANGPTL3
AD-45934.1
68.20
64.11
76.92
79.57
92.11
15.79
11.25
19.99
26.08
26.30
(+) control
AD-19200
41.09
85.94
95.13
101.29
96.60
9.99
25.31
24.56
32.26
26.35
(+) control
AD-19200
23.99
72.76
86.51
108.10
111.13
5.35
34.52
29.24
35.99
31.88
(−) control
AD-1955
89.65
99.87
94.59
104.04
105.10
4.57
5.94
4.19
5.78
7.46
(−) control
AD-1955
104.74
99.78
105.79
109.19
108.08
10.94
7.74
11.12
7.91
10.30
(−) control
mock
100.00
6.92
(−) control
mock
100.00
9.85
(+) control
PLK
10.66
26.65
46.16
92.42
98.78
1.70
8.65
13.47
22.99
23.48
(+) control
PLK
10.74
11.41
17.33
61.02
86.59
3.39
2.61
1.49
27.42
37.31
HeLa day 6
ANGPTL3
AD-15838.2
47.94
80.97
90.44
94.37
96.10
29.05
25.12
13.62
8.88
4.72
ANGPTL3
AD-15838.2
40.32
83.80
89.88
95.94
98.27
22.47
16.51
10.03
3.83
4.19
ANGPTL3
AD-45860.1
57.38
84.84
88.90
96.74
94.03
24.55
17.35
9.67
3.17
6.58
ANGPTL3
AD-45864.1
98.65
100.87
101.13
96.86
98.24
4.35
1.91
2.22
3.41
1.80
ANGPTL3
AD-45870.1
92.69
98.71
98.49
100.07
99.28
3.94
2.67
2.36
1.19
2.65
ANGPTL3
AD-45873.1
91.78
97.38
98.81
97.57
96.22
12.47
6.26
4.08
6.22
8.64
ANGPTL3
AD-45876.1
63.54
85.68
92.13
96.48
95.97
14.74
16.50
10.03
5.81
7.51
ANGPTL3
AD-45877.1
94.17
93.21
96.39
96.70
96.98
7.12
8.00
4.58
3.05
6.15
ANGPTL3
AD-45878.1
66.46
85.75
89.73
94.60
96.59
8.20
7.41
5.27
3.21
3.91
ANGPTL3
AD-45878.1
70.80
89.30
92.54
96.60
95.09
5.18
2.13
1.61
0.50
4.15
ANGPTL3
AD-45879.1
8.29
48.25
73.54
87.47
92.19
4.66
20.05
16.04
9.06
7.90
ANGPTL3
AD-45886.1
23.69
60.65
78.49
93.41
94.15
8.19
13.90
7.15
3.35
4.06
ANGPTL3
AD-45887.1
7.24
26.03
57.68
95.99
98.80
3.07
13.10
14.94
1.40
2.54
ANGPTL3
AD-45915.1
10.38
58.38
85.69
97.24
99.76
6.83
15.66
8.39
1.33
4.15
ANGPTL3
AD-45929.1
11.73
36.67
51.90
76.71
85.08
4.80
14.19
15.34
12.37
10.60
ANGPTL3
AD-45934.1
73.57
88.48
92.94
91.50
95.97
5.36
2.96
5.50
5.44
4.39
(+) control
AD-19200
63.58
90.14
95.44
94.65
93.28
34.11
14.32
8.78
10.90
12.13
(+) control
AD-19200
16.05
78.65
85.78
93.09
96.22
9.77
15.57
19.50
13.34
10.96
(−) control
AD-1955
93.52
97.36
97.90
99.65
100.07
5.02
1.78
0.84
0.58
1.14
(−) control
AD-1955
75.39
93.61
97.79
99.60
100.96
8.37
2.50
2.27
2.68
3.16
(−) control
mock
100.00
1.32
(−) control
mock
100.00
3.35
(+) control
PLK
3.68
55.22
63.00
89.39
95.33
1.42
30.96
33.97
15.85
8.54
(+) control
PLK
2.69
3.74
9.74
67.07
82.96
0.15
0.96
3.60
22.70
19.34
Hep3B day 3
ANGPTL3
AD-15838.2
35.33
61.00
68.79
82.74
90.41
2.41
6.21
4.21
2.61
7.07
ANGPTL3
AD-15838.2
35.34
61.04
72.14
89.71
106.88
1.49
2.61
7.37
6.48
7.13
ANGPTL3
AD-45860.1
17.79
39.25
60.57
94.28
99.85
1.07
3.51
3.57
13.09
16.41
ANGPTL3
AD-45864.1
80.35
88.19
87.01
89.39
92.09
6.93
6.98
9.42
7.41
17.05
ANGPTL3
AD-45870.1
75.00
93.30
96.64
106.29
99.08
7.10
12.24
4.01
5.95
9.64
ANGPTL3
AD-45873.1
42.68
78.45
82.26
97.11
96.58
5.17
5.04
8.31
12.11
11.33
ANGPTL3
AD-45876.1
31.37
55.00
70.69
93.49
91.00
4.39
6.09
5.47
15.11
6.38
ANGPTL3
AD-45877.1
74.45
94.60
96.70
103.77
106.75
3.27
2.44
3.45
6.10
7.40
ANGPTL3
AD-45878.1
50.22
69.65
80.49
92.77
97.37
2.51
14.94
10.44
8.21
5.30
ANGPTL3
AD-45878.1
44.85
65.39
75.67
92.83
109.67
10.10
7.76
8.56
7.78
4.97
ANGPTL3
AD-45879.1
23.73
60.81
84.59
95.72
108.68
6.43
21.36
19.62
13.69
5.95
ANGPTL3
AD-45886.1
27.19
55.35
64.97
100.18
102.09
0.97
6.65
11.46
6.91
4.08
ANGPTL3
AD-45887.1
41.70
97.18
101.91
111.27
105.18
9.26
6.81
7.36
1.72
2.23
ANGPTL3
AD-45915.1
45.10
66.31
82.22
97.97
103.30
6.91
11.84
14.79
6.54
2.48
ANGPTL3
AD-45929.1
48.58
79.14
89.96
95.00
101.37
10.40
10.29
10.52
18.24
10.53
ANGPTL3
AD-45934.1
80.15
102.93
112.82
114.16
113.98
5.28
0.62
4.19
0.75
3.99
(+) control
AD-19200
14.79
55.23
72.90
89.64
94.30
2.17
5.42
7.19
10.28
16.39
(+) control
AD-19200
22.76
92.02
101.56
106.68
113.09
6.61
18.99
7.41
9.83
10.64
(−) control
AD-1955
77.77
81.25
82.23
88.21
95.02
2.83
5.40
5.08
5.42
6.63
(−) control
AD-1955
80.42
86.70
90.23
93.46
97.04
10.53
5.70
8.14
3.27
3.45
(−) control
mock
100.00
5.77
(−) control
mock
100.00
9.79
(+) control
PLK
10.91
12.89
14.31
23.87
50.93
0.17
0.87
1.64
1.13
7.80
(+) control
PLK
13.19
16.12
22.89
55.03
94.35
0.78
0.88
8.36
18.88
9.85
Hep3B day 6
ANGPTL3
AD-15838.2
78.88
89.58
93.08
91.10
100.66
11.60
9.15
12.04
10.51
5.87
ANGPTL3
AD-15838.2
81.17
85.91
87.27
103.95
103.59
7.75
3.29
8.07
7.93
9.82
ANGPTL3
AD-45860.1
84.11
87.77
93.22
99.15
96.75
14.22
13.36
20.98
13.15
17.62
ANGPTL3
AD-45864.1
99.27
111.82
106.28
99.15
97.55
7.77
16.31
14.24
15.40
9.18
ANGPTL3
AD-45870.1
95.49
109.60
104.16
104.65
106.76
11.92
12.98
9.25
10.29
19.12
ANGPTL3
AD-45873.1
71.45
90.62
93.44
102.07
107.72
4.71
4.40
15.02
11.96
10.16
ANGPTL3
AD-45876.1
76.92
82.09
89.44
95.27
105.41
9.39
13.55
7.93
9.77
10.42
ANGPTL3
AD-45877.1
82.98
98.05
95.07
103.55
104.14
11.22
13.45
1.27
8.88
6.49
ANGPTL3
AD-45878.1
75.14
82.48
89.68
92.71
95.72
8.65
10.07
10.77
12.44
15.04
ANGPTL3
AD-45878.1
65.90
77.37
78.33
84.54
99.49
10.21
13.22
9.95
11.65
11.17
ANGPTL3
AD-45879.1
86.42
89.45
101.50
97.30
100.66
10.59
10.12
19.77
13.19
9.54
ANGPTL3
AD-45886.1
91.15
79.31
80.76
86.52
94.04
12.89
11.88
5.38
4.92
6.80
ANGPTL3
AD-45887.1
91.67
103.38
107.88
100.05
102.05
10.80
14.84
19.18
13.72
18.00
ANGPTL3
AD-45915.1
81.97
85.91
91.81
94.95
102.13
18.49
19.30
7.19
12.72
16.64
ANGPTL3
AD-45929.1
61.92
79.39
87.28
88.09
96.00
6.80
10.76
5.80
10.68
16.66
ANGPTL3
AD-45934.1
85.84
89.66
97.67
99.91
102.54
12.39
14.25
4.74
9.51
4.28
(+) control
AD-19200
50.48
65.62
79.67
98.61
96.87
4.60
4.64
7.20
5.08
7.37
(+) control
AD-19200
52.01
75.89
92.59
101.47
99.66
4.35
20.87
13.57
6.50
11.76
(−) control
AD-1955
91.77
95.87
93.06
95.10
97.52
8.87
3.46
1.46
2.00
3.84
(−) control
AD-1955
93.65
94.41
89.42
100.59
103.91
9.91
14.90
6.80
11.99
10.31
(−) control
mock
100.00
5.10
(−) control
mock
100.00
7.35
(+) control
PLK
36.43
37.75
40.19
55.25
64.59
3.44
2.75
3.65
5.33
5.02
(+) control
PLK
38.70
43.68
50.32
75.17
89.62
3.40
3.85
8.10
10.54
10.69
TABLE 7
Unmodified sense and antisense strand sequences of ANGPTL3 GalNac-conjugated dsRNAs
Sense Sequence
(SEQ ID NOS 264-448,
Antisense Sequence
respectively,
(SEQ ID NOS 449-633,
Sense
in order of
Position in
Antisense
respectively,
Position in
Duplex ID
Name
appearance)
NM_014495.2 _
Name
in order of appearance)
NM_014495.2
AD-53063.1
A-108558.1
AAAGACAACAAACAUUAUAUUx
1066-1086
A-108559.1
AAUAUAAUGUUUGUUGUCUUUCC
1064-1086
AD-52965.1
A-108310.1
ACAAUUAAGCUCCUUCUUUUUx
58-78
A-108311.1
AAAAAGAAGGAGCUUAAUUGUGA
56-78
AD-53030.1
A-108410.1
UGUCACUUGAACUCAACUCAAx
398-418
A-108411.1
UUGAGUUGAGUUCAAGUGACAUA
396-418
AD-52953.1
A-108306.1
UCACAAUUAAGCUCCUUCUUUx
56-76
A-108307.1
AAAGAAGGAGCUUAAUUGUGAAC
54-76
AD-53001.1
A-108416.1
CUUGAACUCAACUCAAAACUUx
403-423
A-108417.1
AAGUUUUGAGUUGAGUUCAAGUG
401-423
AD-53080.1
A-108548.1
CUCCAUAGUGAAGCAAUCUAAx
1014-1034
A-108549.1
UUAGAUUGCUUCACUAUGGAGUA
1012-1034
AD-52971.1
A-108312.1
CAAUUAAGCUCCUUCUUUUUAx
59-79
A-108313.1
UAAAAAGAAGGAGCUUAAUUGUG
57-79
AD-53071.1
A-108498.1
ACCCAGCAACUCUCAAGUUUUx
840-860
A-108499.1
AAAACUUGAGAGUUGCUGGGUCU
838-860
AD-53024.1
A-108408.1
GAAUAUGUCACUUGAACUCAAx
393-413
A-108409.1
UUGAGUUCAAGUGACAUAUUCUU
391-413
AD-52977.1
A-108314.1
AAUUAAGCUCCUUCUUUUUAUx
60-80
A-108315.1
AUAAAAAGAAGGAGCUUAAUUGU
58-80
AD-53064.1
A-108574.1
CAUUAUAUUGAAUAUUCUUUUx
1078-1098
A-108575.1
AAAAGAAUAUUCAAUAUAAUGUU
1076-1098
AD-53033.1
A-108458.1
ACUAACUAACUUAAUUCAAAAx
483-503
A-108459.1
UUUUGAAUUAAGUUAGUUAGUUG
481-503
AD-52954.1
A-108322.1
UUAUUGUUCCUCUAGUUAUUUx
77-97
A-108323.1
AAAUAACUAGAGGAACAAUAAAA
75-97
AD-53098.1
A-108554.1
CAUAGUGAAGCAAUCUAAUUAx
1017-1037
A-108555.1
UAAUUAGAUUGCUUCACUAUGGA
1015-1037
AD-53092.1
A-108552.1
CCAUAGUGAAGCAAUCUAAUUx
1016-1036
A-108553.1
AAUUAGAUUGCUUCACUAUGGAG
1014-1036
AD-53073.1
A-108530.1
GAUCACAAAACUUCAAUGAAAx
923-943
A-108531.1
UUUCAUUGAAGUUUUGUGAUCCA
921-943
AD-53132.1
A-108628.1
AUGGAAGGUUAUACUCUAUAAx
1364-1384
A-108629.1
UUAUAGAGUAUAACCUUCCAUUU
1362-1384
AD-53086.1
A-108550.1
UCCAUAGUGAAGCAAUCUAAUx
1015-1035
A-108551.1
AUUAGAUUGCUUCACUAUGGAGU
1013-1035
AD-52961.1
A-108340.1
CUAUGUUAGACGAUGUAAAAAx
164-184
A-108341.1
UUUUUACAUCGUCUAACAUAGCA
162-184
AD-52983.1
A-108316.1
AUUAAGCUCCUUCUUUUUAUUx
61-81
A-108317.1
AAUAAAAAGAAGGAGCUUAAUUG
59-81
AD-53027.1
A-108456.1
AACUAACUAACUUAAUUCAAAx
482-502
A-108457.1
UUUGAAUUAAGUUAGUUAGUUGC
480-502
AD-52986.1
A-108364.1
GGCCAAAUUAAUGACAUAUUUx
247-267
A-108365.1
AAAUAUGUCAUUAAUUUGGCCCU
245-267
AD-52989.1
A-108318.1
UUUUAUUGUUCCUCUAGUUAUx
75-95
A-108319.1
AUAACUAGAGGAACAAUAAAAAG
73-95
AD-52981.1
A-108378.1
ACAUAUUUGAUCAGUCUUUUUx
278-298
A-108379.1
AAAAAGACUGAUCAAAUAUGUUG
276-298
AD-53077.1
A-108500.1
CCCAGCAACUCUCAAGUUUUUx
841-861
A-108501.1
AAAAACUUGAGAGUUGCUGGGUC
839-861
AD-53095.1
A-108506.1
CAGGUAGUCCAUGGACAUUAAx
884-904
A-108507.1
UUAAUGUCCAUGGACUACCUGAU
882-904
AD-52970.1
A-108390.1
ACUGAGAAGAACUACAUAUAAx
345-365
A-108391.1
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UUGAAUUAAUGUCCAUGGACUAC
887-909
AD-53110.1
A-108652.1
AACUGAGGCAAAUUUAAAAGAx
1434-1454
A-108653.1
UCUUUUAAAUUUGCCUCAGUUCA
1432-
1454_G21A
AD-52980.1
A-108362.1
GGGCCAAAUUAAUGACAUAUUx
246-266
A-108363.1
AAUAUGUCAUUAAUUUGGCCCUU
244-266
AD-53109.1
A-108636.1
AUCCAUCCAACAGAUUCAGAAx
1402-1422
A-108637.1
UUCUGAAUCUGUUGGAUGGAUCA
1400-1422
AD-53141.1
A-108600.1
AAGAUUUGGUGUUUUCUACUUx
1181-1201
A-108601.1
AAGUAGAAAACACCAAAUCUUUG
1179-1201
AD-53126.1
A-108626.1
GUCUCAAAAUGGAAGGUUAUAx
1356-1376
A-108627.1
UAUAACCUUCCAUUUUGAGACUU
1354-1376
AD-53116.1
A-108654.1
ACUGAGGCAAAUUUAAAAGGAx
1435-1455
A-108655.1
UCCUUUUAAAUUUGCCUCAGUUC
1433-
1455_C21A
AD-52997.1
A-108352.1
GGGACAUGGUCUUAAAGACUUx
210-230
A-108353.1
AAGUCUUUAAGACCAUGUCCCAA
208-230
AD-53120.1
A-108624.1
AUGGUAAAUAUAACAAACCAAx
1292-1312
A-108625.1
UUGGUUUGUUAUAUUUACCAUUU
1290-1312
AD-53070.1
A-108576.1
GGGAAAUCACGAAACCAACUAx
1104-1124
A-108577.1
UAGUUGGUUUCGUGAUUUCCCAA
1102-1124
AD-53028.1
A-108472.1
CCAACAGCAUAGUCAAAUAAAx
621-641
A-108473.1
UUUAUUUGACUAUGCUGUUGGUU
619-641
AD-53146.1
A-108602.1
UUUUCUACUUGGGAUCACAAAx
1192-1212
A-108603.1
UUUGUGAUCCCAAGUAGAAAACA
1190-1212
AD-52982.1
A-108394.1
AGAACUACAUAUAAACUACAAx
352-372
A-108395.1
UUGUAGUUUAUAUGUAGUUCUUC
350-372
AD-53111.1
A-108668.1
AGAGUAUGUGUAAAAAUCUGUx
1915-1935
A-108669.1
ACAGAUUUUUACACAUACUCUGU
1913-1935
AD-53045.1
A-108462.1
AAAACAAGAUAAUAGCAUCAAx
558-578
A-108463.1
UUGAUGCUAUUAUCUUGUUUUUC
556-578
AD-53123.1
A-108672.1
AGUAUGUGUAAAAAUCUGUAAx
1917-1937
A-108673.1
UUACAGAUUUUUACACAUACUCU
1915-1937
AD-53018.1
A-108406.1
AGUCAAAAAUGAAGAGGUAAAx
372-392
A-108407.1
UUUACCUCUUCAUUUUUGACUUG
370-392
AD-52956.1
A-108354.1
GGACAUGGUCUUAAAGACUUUx
211-231
A-108355.1
AAAGUCUUUAAGACCAUGUCCCA
209-231
AD-53134.1
A-108660.1
GAGGCAAAUUUAAAAGGCAAUx
1438-1458
A-108661.1
AUUGCCUUUUAAAUUUGCCUCAG
1436-1458
AD-52968.1
A-108358.1
GUCUUAAAGACUUUGUCCAUAx
218-238
A-108359.1
UAUGGACAAAGUCUUUAAGACCA
216-238
AD-53122.1
A-108656.1
CUGAGGCAAAUUUAAAAGGCAx
1436-1456
A-108657.1
UGCCUUUUAAAUUUGCCUCAGUU
1434-1456
AD-53100.1
A-108586.1
GCAAUCCCGGAAAACAAAGAUx
1165-1185
A-108587.1
AUCUUUGUUUUCCGGGAUUGCAU
1163-1185
AD-53128.1
A-108658.1
UGAGGCAAAUUUAAAAGGCAAx
1437-1457
A-108659.1
UUGCCUUUUAAAUUUGCCUCAGU
1435-1457
AD-53043.1
A-108430.1
UCUACUUCAACAAAAAGUGAAx
447-467
A-108431.1
UUCACUUUUUGUUGAAGUAGAAU
445-467
AD-53135.1
A-108676.1
UAUGUGUAAAAAUCUGUAAUAx
1919-1939
A-108677.1
UAUUACAGAUUUUUACACAUACU
1917-1939
AD-53094.1
A-108584.1
AAUGCAAUCCCGGAAAACAAAx
1162-1182
A-108585.1
UUUGUUUUCCGGGAUUGCAUUGG
1160-1182
AD-53019.1
A-108422.1
CUUGAAAGCCUCCUAGAAGAAx
421-441
A-108423.1
UUCUUCUAGGAGGCUUUCAAGUU
419-441
AD-53129.1
A-108674.1
GUAUGUGUAAAAAUCUGUAAUx
1918-1938
A-108675.1
AUUACAGAUUUUUACACAUACUC
1916-1938
AD-53150.1
A-108666.1
CAGAGUAUGUGUAAAAAUCUUx
1914-1934
A-108667.1
AAGAUUUUUACACAUACUCUGUG
1912-
1934_G21U
AD-53117.1
A-108670.1
GAGUAUGUGUAAAAAUCUGUAx
1916-1936
A-108671.1
UACAGAUUUUUACACAUACUCUG
1914-1936
AD-52985.1
A-108348.1
UCAGUUGGGACAUGGUCUUAAx
204-224
A-108349.1
UUAAGACCAUGUCCCAACUGAAG
202-224
AD-52962.1
A-108356.1
GGUCUUAAAGACUUUGUCCAUx
217-237
A-108357.1
AUGGACAAAGUCUUUAAGACCAU
215-237
AD-52974.1
A-108360.1
UCUUAAAGACUUUGUCCAUAAx
219-239
A-108361.1
UUAUGGACAAAGUCUUUAAGACC
217-239
AD-52979.1
A-108346.1
UUCAGUUGGGACAUGGUCUUAx
203-223
A-108347.1
UAAGACCAUGUCCCAACUGAAGG
201-223
The symbol “x” indicates that the sequence contains a GalNAc conjugate.
TABLE 8
Modified sense and antisense strand sequences of ANGPTL3 GalNac-conjugated dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 634-818,
(SEQ ID NOS 819-1003,
Sense
respectively, in
Antisense
respectively,
Duplex ID
OligoName
order of appearance)
OligoName
in order of appearance)
AD-53063.1
A-108558.1
AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUfL96
A-108559.1
aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc
AD-52965.1
A-108310.1
AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUfL96
A-108311.1
aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa
AD-53030.1
A-108410.1
UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAfL96
A-108411.1
uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa
AD-52953.1
A-108306.1
UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUfL96
A-108307.1
aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc
AD-53001.1
A-108416.1
CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUfL96
A-108417.1
aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg
AD-53080.1
A-108548.1
CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAfL96
A-108549.1
uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa
AD-52971.1
A-108312.1
CfaAfuUfaAfgCfUfCfcUfuCfuUfuUfuAfL96
A-108313.1
uAfaAfaAfgAfaGfgagCfuUfaAfuUfgsUfsg
AD-53071.1
A-108498.1
AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUfL96
A-108499.1
aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu
AD-53024.1
A-108408.1
GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAfL96
A-108409.1
uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu
AD-52977.1
A-108314.1
AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUfL96
A-108315.1
aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu
AD-53064.1
A-108574.1
CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUfL96
A-108575.1
aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu
AD-53033.1
A-108458.1
AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAfL96
A-108459.1
uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg
AD-52954.1
A-108322.1
UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUfL96
A-108323.1
aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa
AD-53098.1
A-108554.1
CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAfL96
A-108555.1
uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa
AD-53092.1
A-108552.1
CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUfL96
A-108553.1
aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg
AD-53073.1
A-108530.1
GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAfL96
A-108531.1
uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa
AD-53132.1
A-108628.1
AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAfL96
A-108629.1
uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu
AD-53086.1
A-108550.1
UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUfL96
A-108551.1
aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu
AD-52961.1
A-108340.1
CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAfL96
A-108341.1
uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa
AD-52983.1
A-108316.1
AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUfL96
A-108317.1
aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg
AD-53027.1
A-108456.1
AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAfL96
A-108457.1
uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc
AD-52986.1
A-108364.1
GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUfL96
A-108365.1
aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu
AD-52989.1
A-108318.1
UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUfL96
A-108319.1
aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg
AD-52981.1
A-108378.1
AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96
A-108379.1
aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg
AD-53077.1
A-108500.1
CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUfL96
A-108501.1
aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc
AD-53095.1
A-108506.1
CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAfL96
A-108507.1
uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu
AD-52970.1
A-108390.1
AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAfL96
A-108391.1
uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc
AD-53015.1
A-108452.1
GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUfL96
A-108453.1
aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu
AD-53147.1
A-108618.1
AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUfL96
A-108619.1
aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu
AD-53103.1
A-108540.1
CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUfL96
A-108541.1
aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc
AD-52969.1
A-108374.1
CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUfL96
A-108375.1
aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg
AD-53075.1
A-108562.1
AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUfL96
A-108563.1
aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu
AD-52994.1
A-108398.1
AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAfL96
A-108399.1
uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg
AD-52960.1
A-108324.1
CfuAfgUfuAfuUfUfCfcUfcCfaGfaAfuUfL96
A-108325.1
aAfuUfcUfgGfaGfgaaAfuAfaCfuAfgsAfsg
AD-53003.1
A-108448.1
AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAfL96
A-108449.1
uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu
AD-52995.1
A-108320.1
UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUfL96
A-108321.1
aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa
AD-53037.1
A-108428.1
CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAfL96
A-108429.1
uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc
AD-53087.1
A-108566.1
AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUfL96
A-108567.1
aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu
AD-53076.1
A-108578.1
GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUfL96
A-108579.1
aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa
AD-52975.1
A-108376.1
AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUfL96
A-108377.1
aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa
AD-53138.1
A-108630.1
UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAfL96
A-108631.1
uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu
AD-53091.1
A-108536.1
GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUfL96
A-108537.1
aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa
AD-53124.1
A-108594.1
GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUfL96
A-108595.1
aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg
AD-53125.1
A-108610.1
AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAfL96
A-108611.1
uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa
AD-53036.1
A-108412.1
GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAfL96
A-108413.1
uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu
AD-53061.1
A-108526.1
GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUfL96
A-108527.1
aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa
AD-53093.1
A-108568.1
AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUfL96
A-108569.1
aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg
AD-53137.1
A-108614.1
UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUfL96
A-108615.1
aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu
AD-52999.1
A-108384.1
AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUfL96
A-108385.1
aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa
AD-53069.1
A-108560.1
GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAfL96
A-108561.1
uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu
AD-53034.1
A-108474.1
CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAfL96
A-108475.1
uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu
AD-52976.1
A-108392.1
CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAfL96
A-108393.1
uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu
AD-52996.1
A-108336.1
UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAfL96
A-108337.1
uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa
AD-53029.1
A-108488.1
AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUfL96
A-108489.1
aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu
AD-53020.1
A-108438.1
CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUfL96
A-108439.1
aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa
AD-53042.1
A-108414.1
UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAfL96
A-108415.1
uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa
AD-53011.1
A-108482.1
CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAfL96
A-108483.1
uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu
AD-52957.1
A-108370.1
CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUfL96
A-108371.1
aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa
AD-53008.1
A-108434.1
UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUfL96
A-108435.1
aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa
AD-53065.1
A-108496.1
GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUfL96
A-108497.1
aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg
AD-53115.1
A-108638.1
UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUfL96
A-108639.1
aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg
AD-53012.1
A-108404.1
UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUfL96
A-108405.1
aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg
AD-53004.1
A-108464.1
AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAfL96
A-108465.1
uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu
AD-53021.1
A-108454.1
CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAfL96
A-108455.1
uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu
AD-52955.1
A-108338.1
GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAfL96
A-108339.1
uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa
AD-53119.1
A-108608.1
AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAfL96
A-108609.1
uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg
AD-52990.1
A-108334.1
UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAfL96
A-108335.1
uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu
AD-52964.1
A-108388.1
AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAfL96
A-108389.1
uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc
AD-52973.1
A-108344.1
GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUfL96
A-108345.1
aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu
AD-53074.1
A-108546.1
AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAfL96
A-108547.1
uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu
AD-53026.1
A-108440.1
UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUfL96
A-108441.1
aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu
AD-53062.1
A-108542.1
CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAfL96
A-108543.1
uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc
AD-53114.1
A-108622.1
CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAfL96
A-108623.1
uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu
AD-53082.1
A-108580.1
GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAfL96
A-108581.1
uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc
AD-53035.1
A-108490.1
CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUfL96
A-108491.1
aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu
AD-52978.1
A-108330.1
AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAfL96
A-108331.1
uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg
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AD-52985.1
A-108348.1
UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAfL96
A-108349.1
uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg
AD-52962.1
A-108356.1
GfgUfcUfuAfaAfGfAfcUfuUfgUfcCfaUfL96
A-108357.1
aUfgGfaCfaAfaGfucuUfuAfaGfaCfcsAfsu
AD-52974.1
A-108360.1
UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAfL96
A-108361.1
uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc
AD-52979.1
A-108346.1
UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAfL96
A-108347.1
uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides;
Nf (e.g., Af) is a 2′-fluoro nucleotide;
s is a phosphothiorate linkage;
L96 indicates a GalNAc ligand.
TABLE 9
Unmodified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences are the same as the sequences
listed in Table 7 except that they do not
contain GalNal conjugation.
Anti-
Sense
sense
Se-
Se-
quence
quence
(SEQ
(SEQ
ID
ID
NOS
NOS
1004-
1185-
1184,
1365,
respec-
respec-
tively,
tively,
in
in
Posi-
order
Anti-
order
tion
Sense
of
sense
of
in
Duplex
Oligo
appear-
Oligo
appear-
NM_014
Name
Name
ance)
Name
ance)
495.2
AD-
A-
UCAC
A-
AAAG
54-76
52637.1
108817.1
AAUU
108307.2
AAGG
AAGC
AGCU
UCCU
UAAU
UCUU
UGUG
U
AAC
AD-
A-
UUAU
A-
AAAU
75-97
52638.1
108825.1
UGUU
108323.2
AACU
CCUC
AGAG
UAGU
GAAC
UAUU
AAUA
U
AAA
AD-
A-
GCUA
A-
UUUU
161-183
52639.1
108833.1
UGUU
108339.2
ACAU
AGAC
CGUC
GAUG
UAAC
UAAA
AUAG
A
CAA
AD-
A-
GGAC
A-
AAAG
209-231
52640.1
108841.1
AUGG
108355.2
UCUU
UCUU
UAAG
AAAG
ACCA
ACUU
UGUC
U
CCA
AD-
A-
CAAA
A-
AUCA
266-288
52641.1
108849.1
AACU
108371.2
AAUA
CAAC
UGUU
AUAU
GAGU
UUGA
UUUU
U
GAA
AD-
A-
ACCA
A-
UUCU
314-336
52642.1
108857.1
GUGA
108387.2
UCUU
AAUC
UGAU
AAAG
UUCA
AAGA
CUGG
A
UUU
AD-
A-
CACA
A-
AAAA
55-77
52643.1
108818.1
AUUA
108309.2
GAAG
AGCU
GAGC
CCUU
UUAA
CUUU
UUGU
U
GAA
AD-
A-
CUAU
A-
UUUU
162-184
52645.1
108834.1
GUUA
108341.2
UACA
GACG
UCGU
AUGU
CUAA
AAAA
CAUA
A
GCA
AD-
A-
UCAA
A-
AAGA
273-295
52647.1
108850.1
CAUA
108373.2
CUGA
UUUG
UCAA
AUCA
AUAU
GUCU
GUUG
U
AGU
AD-
A-
AACU
A-
UAUA
342-364
52648.1
108858.1
GAGA
108389.2
UGUA
AGAA
GUUC
CUAC
UUCU
AUAU
CAGU
A
UCC
AD-
A-
ACAA
A-
AAAA
56-78
52649.1
108819.1
UUAA
108311.2
AGAA
GCUC
GGAG
CUUC
CUUA
UUUU
AUUG
U
UGA
AD-
A-
CUCC
A-
AUCU
138-160
52650.1
108827.1
AGAG
108327.2
UGAU
CCAA
UUUG
AAUC
GCUC
AAGA
UGGA
U
GAU
AD-
A-
CGAU
A-
UUGG
172-194
52651.1
108835.1
GUAA
108343.2
CUAA
AAAU
AAUU
UUUA
UUUA
GCCA
CAUC
A
GUC
AD-
A-
GUCU
A-
UAUG
216-238
52652.1
108843.1
UAAA
108359.2
GACA
GACU
AAGU
UUGU
CUUU
CCAU
AAGA
A
CCA
AD-
A-
CAAC
A-
AAAG
274-296
52653.1
108851.1
AUAU
108375.2
ACUG
UUGA
AUCA
UCAG
AAUA
UCUU
UGUU
U
GAG
AD-
A-
ACUG
A-
UUAU
343-365
52654.1
108859.1
AGAA
108391.2
AUGU
GAAC
AGUU
UACA
CUUC
UAUA
UCAG
A
UUC
AD-
A-
CCAG
A-
AAAU
140-162
52656.1
108828.1
AGCC
108329.2
CUUG
AAAA
AUUU
UCAA
UGGC
GAUU
UCUG
U
GAG
AD-
A-
GAUG
A-
AUUG
173-195
52657.1
108836.1
UAAA
108345.2
GCUA
AAUU
AAAU
UUAG
UUUU
CCAA
ACAU
U
CGU
AD-
A-
UCUU
A-
UUAU
217-239
52658.1
108844.1
AAAG
108361.2
GGAC
ACUU
AAAG
UGUC
UCUU
CAUA
UAAG
A
ACC
AD-
A-
AACA
A-
AAAA
275-297
52659.1
108852.1
UAUU
108377.2
GACU
UGAU
GAUC
CAGU
AAAU
CUUU
AUGU
U
UGA
AD-
A-
CUGA
A-
UUUA
344-366
52660.1
108860.1
GAAG
108393.2
UAUG
AACU
UAGU
ACAU
UCUU
AUAA
CUCA
A
GUU
AD-
A-
AAUU
A-
AUAA
58-80
52661.1
108821.1
AAGC
108315.2
AAAG
UCCU
AAGG
UCUU
AGCU
UUUA
UAAU
U
UGU
AD-
A-
AAAU
A-
UAAC
149-171
52662.1
108829.1
CAAG
108331.2
AUAG
AUUU
CAAA
GCUA
UCUU
UGUU
GAUU
A
UUG
AD-
A-
UUCA
A-
UAAG
201-223
52663.1
108837.1
GUUG
108347.2
ACCA
GGAC
UGUC
AUGG
CCAA
UCUU
CUGA
A
AGG
AD-
A-
GGGC
A-
AAUA
244-266
52664.1
108845.1
CAAA
108363.2
UGUC
UUAA
AUUA
UGAC
AUUU
AUAU
GGCC
U
CUU
AD-
A-
ACAU
A-
AAAA
276-298
52665.1
108853.1
AUUU
108379.2
AGAC
GAUC
UGAU
AGUC
CAAA
UUUU
UAUG
U
UUG
AD-
A-
AGAA
A-
UUGU
350-372
52666.1
108861.1
CUAC
108395.2
AGUU
AUAU
UAUA
AAAC
UGUA
UACA
GUUC
A
UUC
AD-
A-
AUUA
A-
AAUA
59-81
52667.1
108822.1
AGCU
108317.2
AAAA
CCUU
GAAG
CUUU
GAGC
UUAU
UUAA
U
UUG
AD-
A-
AGAU
A-
AUCG
155-177
52668.1
108830.1
UUGC
108333.2
UCUA
UAUG
ACAU
UUAG
AGCA
ACGA
AAUC
U
UUG
AD-
A-
UCAG
A-
UUAA
202-224
52669.1
108838.1
UUGG
108349.2
GACC
GACA
AUGU
UGGU
CCCA
CUUA
ACUG
A
AAG
AD-
A-
GGCC
A-
AAAU
245-267
52670.1
108846.1
AAAU
108365.2
AUGU
UAAU
CAUU
GACA
AAUU
UAUU
UGGC
U
CCU
AD-
A-
CAUA
A-
UAAA
277-299
52671.1
108854.1
UUUG
108381.2
AAGA
AUCA
CUGA
GUCU
UCAA
UUUU
AUAU
A
GUU
AD-
A-
UACA
A-
UUGA
355-377
52672.1
108862.1
UAUA
108397.2
CUUG
AACU
UAGU
ACAA
UUAU
GUCA
AUGU
A
AGU
AD-
A-
UUUU
A-
AUAA
73-95
52673.1
108823.1
AUUG
108319.2
CUAG
UUCC
AGGA
UCUA
ACAA
GUUA
UAAA
U
AAG
AD-
A-
UUGC
A-
UUAC
159-181
52674.1
108831.1
UAUG
108335.2
AUCG
UUAG
UCUA
ACGA
ACAU
UGUA
AGCA
A
AAU
AD-
A-
CAGU
A-
UUUA
203-225
52675.1
108839.1
UGGG
108351.2
AGAC
ACAU
CAUG
GGUC
UCCC
UUAA
AACU
A
GAA
AD-
A-
AAAU
A-
UUUG
249-271
52676.1
108847.1
UAAU
108367.2
AAAU
GACA
AUGU
UAUU
CAUU
UCAA
AAUU
A
UGG
AD-
A-
GAUC
A-
UAGA
284-306
52677.1
108855.1
AGUC
108383.2
UCAU
UUUU
AAAA
UAUG
AGAC
AUCU
UGAU
A
CAA
AD-
A-
ACAU
A-
UUUG
356-378
52678.1
108863.1
AUAA
108399.2
ACUU
ACUA
GUAG
CAAG
UUUA
UCAA
UAUG
A
UAG
AD-
A-
UUUA
A-
AAUA
74-96
52679.1
108824.1
UUGU
108321.2
ACUA
UCCU
GAGG
CUAG
AACA
UUAU
AUAA
U
AAA
AD-
A-
UGCU
A-
UUUA
160-182
52680.1
108832.1
AUGU
108337.2
CAUC
UAGA
GUCU
CGAU
AACA
GUAA
UAGC
A
AAA
AD-
A-
GGGA
A-
AAGU
208-230
52681.1
108840.1
CAUG
108353.2
CUUU
GUCU
AAGA
UAAA
CCAU
GACU
GUCC
U
CAA
AD-
A-
UGAC
A-
UUGA
256-278
52682.1
108848.1
AUAU
108369.2
GUUU
UUCA
UUGA
AAAA
AAUA
CUCA
UGUC
A
AUU
AD-
A-
AUCA
A-
AUAG
285-307
52683.1
108856.1
GUCU
108385.2
AUCA
UUUU
UAAA
AUGA
AAGA
UCUA
CUGA
U
UCA
AD-
A-
CAUA
A-
UUUU
357-379
52684.1
108864.1
UAAA
108401.2
GACU
CUAC
UGUA
AAGU
GUUU
CAAA
AUAU
A
GUA
AD-
A-
CUUG
A-
AAGU
401-423
52685.1
108872.1
AACU
108417.2
UUUG
CAAC
AGUU
UCAA
GAGU
AACU
UCAA
U
GUG
AD-
A-
CUAC
A-
UUUC
446-468
52686.1
108880.1
UUCA
108433.2
ACUU
ACAA
UUUG
AAAG
UUGA
UGAA
AGUA
A
GAA
AD-
A-
AAGA
A-
UUAA
474-496
52687.1
108888.1
GCAA
108449.2
GUUA
CUAA
GUUA
CUAA
GUUG
CUUA
CUCU
A
UCU
AD-
A-
AAAC
A-
UUUG
557-579
52688.1
108896.1
AAGA
108465.2
AUGC
UAAU
UAUU
AGCA
AUCU
UCAA
UGUU
A
UUU
AD-
A-
GCAU
A-
AUUU
625-647
52689.1
108904.1
AGUC
108481.2
CUUU
AAAU
UAUU
AAAA
UGAC
GAAA
UAUG
U
CUG
AD-
A-
AUAU
A-
UUUU
358-380
52690.1
108865.1
AAAC
108403.2
UGAC
UACA
UUGU
AGUC
AGUU
AAAA
UAUA
A
UGU
AD-
A-
GAAC
A-
UUCA
404-426
52691.1
108873.1
UCAA
108419.2
AGUU
CUCA
UUGA
AAAC
GUUG
UUGA
AGUU
A
CAA
AD-
A-
UACU
A-
AUUU
447-469
52692.1
108881.1
UCAA
108435.2
CACU
CAAA
UUUU
AAGU
GUUG
GAAA
AAGU
U
AGA
AD-
A-
AGAG
A-
AUUA
475-497
52693.1
108889.1
CAAC
108451.2
AGUU
UAAC
AGUU
UAAC
AGUU
UUAA
GCUC
U
UUC
AD-
A-
GAUA
A-
AAGG
563-585
52694.1
108897.1
AUAG
108467.2
UCUU
CAUC
UGAU
AAAG
GCUA
ACCU
UUAU
U
CUU
AD-
A-
CAUA
A-
UAUU
626-648
52695.1
108905.1
GUCA
108483.2
UCUU
AAUA
UUAU
AAAG
UUGA
AAAU
CUAU
A
GCU
AD-
A-
UAUA
A-
AUUU
359-381
52696.1
108866.1
AACU
108405.2
UUGA
ACAA
CUUG
GUCA
UAGU
AAAA
UUAU
U
AUG
AD-
A-
AACU
A-
UUUC
405-427
52697.1
108874.1
CAAC
108421.2
AAGU
UCAA
UUUG
AACU
AGUU
UGAA
GAGU
A
UCA
AD-
A-
ACUU
A-
UAUU
448-470
52698.1
108882.1
CAAC
108437.2
UCAC
AAAA
UUUU
AGUG
UGUU
AAAU
GAAG
A
UAG
AD-
A-
GAGC
A-
AAUU
476-498
52699.1
108890.1
AACU
108453.2
AAGU
AACU
UAGU
AACU
UAGU
UAAU
UGCU
U
CUU
AD-
A-
AACC
A-
UAUU
617-639
52700.1
108898.1
AACA
108469.2
UGAC
GCAU
UAUG
AGUC
CUGU
AAAU
UGGU
A
UUA
AD-
A-
AGUC
A-
UUCU
629-651
52701.1
108906.1
AAAU
108485.2
AUUU
AAAA
CUUU
GAAA
UAUU
UAGA
UGAC
A
UAU
AD-
A-
AGUC
A-
UUUA
370-392
52702.1
108867.1
AAAA
108407.2
CCUC
AUGA
UUCA
AGAG
UUUU
GUAA
UGAC
A
UUG
AD-
A-
CUUG
A-
UUCU
419-441
52703.1
108875.1
AAAG
108423.2
UCUA
CCUC
GGAG
CUAG
GCUU
AAGA
UCAA
A
GUU
AD-
A-
CUUC
A-
AUAU
449-471
52704.1
108883.1
AACA
108439.2
UUCA
AAAA
CUUU
GUGA
UUGU
AAUA
UGAA
U
GUA
AD-
A-
CAAC
A-
UUGA
479-501
52705.1
108891.1
UAAC
108455.2
AUUA
UAAC
AGUU
UUAA
AGUU
UUCA
AGUU
A
GCU
AD-
A-
ACCA
A-
UUAU
618-640
52706.1
108899.1
ACAG
108471.2
UUGA
CAUA
CUAU
GUCA
GCUG
AAUA
UUGG
A
UUU
AD-
A-
GAAC
A-
UAGA
677-699
52707.1
108907.1
CCAC
108487.2
GAAA
AGAA
UUUC
AUUU
UGUG
CUCU
GGUU
A
CUU
AD-
A-
GAAU
A-
UUGA
391-413
52708.1
108868.1
AUGU
108409.2
GUUC
CACU
AAGU
UGAA
GACA
CUCA
UAUU
A
CUU
AD-
A-
UGAA
A-
UUUU
421-443
52709.1
108876.1
AGCC
108425.2
CUUC
UCCU
UAGG
AGAA
AGGC
GAAA
UUUC
A
AAG
AD-
A-
UUCA
A-
AAUA
450-472
52710.1
108884.1
ACAA
108441.2
UUUC
AAAG
ACUU
UGAA
UUUG
AUAU
UUGA
U
AGU
AD-
A-
AACU
A-
UUUG
480-502
52711.1
108892.1
AACU
108457.2
AAUU
AACU
AAGU
UAAU
UAGU
UCAA
UAGU
A
UGC
AD-
A-
CCAA
A-
UUUA
619-641
52712.1
108900.1
CAGC
108473.2
UUUG
AUAG
ACUA
UCAA
UGCU
AUAA
GUUG
A
GUU
AD-
A-
AACC
A-
AUAG
678-700
52713.1
108908.1
CACA
108489.2
AGAA
GAAA
AUUU
UUUC
CUGU
UCUA
GGGU
U
UCU
AD-
A-
UGUC
A-
UUGA
396-418
52714.1
108869.1
ACUU
108411.2
GUUG
GAAC
AGUU
UCAA
CAAG
CUCA
UGAC
A
AUA
AD-
A-
GAAA
A-
UUUU
422-444
52715.1
108877.1
GCCU
108427.2
UCUU
CCUA
CUAG
GAAG
GAGG
AAAA
CUUU
A
CAA
AD-
A-
AAUA
A-
UUAG
465-487
52716.1
108885.1
UUUA
108443.2
UUGC
GAAG
UCUU
AGCA
CUAA
ACUA
AUAU
A
UUC
AD-
A-
ACUA
A-
UUUU
481-503
52717.1
108893.1
ACUA
108459.2
GAAU
ACUU
UAAG
AAUU
UUAG
CAAA
UUAG
A
UUG
AD-
A-
CAAC
A-
UUUU
620-642
52718.1
108901.1
AGCA
108475.2
AUUU
UAGU
GACU
CAAA
AUGC
UAAA
UGUU
A
GGU
AD-
A-
CCAC
A-
AAGA
681-703
52719.1
108909.1
AGAA
108491.2
UAGA
AUUU
GAAA
CUCU
UUUC
AUCU
UGUG
U
GGU
AD-
A-
GUCA
A-
UUUG
397-419
52720.1
108870.1
CUUG
108413.2
AGUU
AACU
GAGU
CAAC
UCAA
UCAA
GUGA
A
CAU
AD-
A-
CUCC
A-
UAGA
428-450
52721.1
108878.1
UAGA
108429.2
AUUU
AGAA
UUUC
AAAA
UUCU
UUCU
AGGA
A
GGC
AD-
A-
AUUU
A-
UAGU
468-490
52722.1
108886.1
AGAA
108445.2
UAGU
GAGC
UGCU
AACU
CUUC
AACU
UAAA
A
UAU
AD-
A-
CUAA
A-
AUUU
482-504
52723.1
108894.1
CUAA
108461.2
UGAA
CUUA
UUAA
AUUC
GUUA
AAAA
GUUA
U
GUU
AD-
A-
CAGC
A-
UUCU
623-645
52724.1
108902.1
AUAG
108477.2
UUUA
UCAA
UUUG
AUAA
ACUA
AAGA
UGCU
A
GUU
AD-
A-
GAAA
A-
AUGU
746-768
52725.1
108910.1
UAAG
108493.2
UUUA
AAAU
CAUU
GUAA
UCUU
AACA
AUUU
U
CAU
AD-
A-
UCAC
A-
UUUU
398-420
52726.1
108871.1
UUGA
108415.2
GAGU
ACUC
UGAG
AACU
UUCA
CAAA
AGUG
A
ACA
AD-
A-
UCUA
A-
UUCA
445-467
52727.1
108879.1
CUUC
108431.2
CUUU
AACA
UUGU
AAAA
UGAA
GUGA
GUAG
A
AAU
AD-
A-
UUUA
A-
UUAG
469-491
52728.1
108887.1
GAAG
108447.2
UUAG
AGCA
UUGC
ACUA
UCUU
ACUA
CUAA
A
AUA
AD-
A-
AAAA
A-
UUGA
556-578
52729.1
108895.1
CAAG
108463.2
UGCU
AUAA
AUUA
UAGC
UCUU
AUCA
GUUU
A
UUC
AD-
A-
AGCA
A-
UUUC
624-646
52730.1
108903.1
UAGU
108479.2
UUUU
CAAA
AUUU
UAAA
GACU
AGAA
AUGC
A
UGU
AD-
A-
AGAC
A-
AACU
836-858
52731.1
108958.1
CCAG
108495.2
UGAG
CAAC
AGUU
UCUC
GCUG
AAGU
GGUC
U
UGA
AD-
A-
AGUC
A-
UUGA
887-909
52732.1
108966.1
CAUG
108511.2
AUUA
GACA
AUGU
UUAA
CCAU
UUCA
GGAC
A
UAC
AD-
A-
GAUG
A-
AUUG
917-939
52733.1
108974.1
GAUC
108527.2
AAGU
ACAA
UUUG
AACU
UGAU
UCAA
CCAU
U
CUA
AD-
A-
CUAG
A-
UAUG
998-
52734.1
108982.1
AGAA
108543.2
GAGU
1020
GAUA
AUAU
UACU
CUUC
CCAU
UCUA
A
GGC
AD-
A-
AAAG
A-
AAUA
1064-
52735.1
108990.1
ACAA
108559.2
UAAU
1086
CAAA
GUUU
CAUU
GUUG
AUAU
UCUU
U
UCC
AD-
A-
CAUU
A-
AAAA
1076-
52736.1
108998.1
AUAU
108575.2
GAAU
1098
UGAA
AUUC
UAUU
AAUA
CUUU
UAAU
U
GUU
AD-
A-
GACC
A-
AAAC
837-859
52737.1
108959.1
CAGC
108497.2
UUGA
AACU
GAGU
CUCA
UGCU
AGUU
GGGU
U
CUG
AD-
A-
GGAU
A-
UUCA
920-942
52739.1
108975.1
CACA
108529.2
UUGA
AAAC
AGUU
UUCA
UUGU
AUGA
GAUC
A
CAU
AD-
A-
GAAG
A-
UUCA
1003-
52740.1
108983.1
AUAU
108545.2
CUAU
1025
ACUC
GGAG
CAUA
UAUA
GUGA
UCUU
A
CUC
AD-
A-
GACA
A-
UUCA
1067-
52741.1
108991.1
ACAA
108561.2
AUAU
1089
ACAU
AAUG
UAUA
UUUG
UUGA
UUGU
A
CUU
AD-
A-
GGGA
A-
UAGU
1102-
52742.1
108999.1
AAUC
108577.2
UGGU
1124
ACGA
UUCG
AACC
UGAU
AACU
UUCC
A
CAA
AD-
A-
ACCC
A-
AAAA
838-860
52743.1
108960.1
AGCA
108499.2
CUUG
ACUC
AGAG
UCAA
UUGC
GUUU
UGGG
U
UCU
AD-
A-
GGAC
A-
UUCG
894-916
52744.1
108968.1
AUUA
108515.2
AUGU
AUUC
UGAA
AACA
UUAA
UCGA
UGUC
A
CAU
AD-
A-
GAUC
A-
UUUC
921-943
52745.1
108976.1
ACAA
108531.2
AUUG
AACU
AAGU
UCAA
UUUG
UGAA
UGAU
A
CCA
AD-
A-
ACUC
A-
UAGA
1011-
52746.1
108984.1
CAUA
108547.2
UUGC
1033
GUGA
UUCA
AGCA
CUAU
AUCU
GGAG
A
UAU
AD-
A-
ACAA
A-
AUUC
1068-
52747.1
108992.1
CAAA
108563.2
AAUA
1090
CAUU
UAAU
AUAU
GUUU
UGAA
GUUG
U
UCU
AD-
A-
GGAA
A-
AUAG
1103-
52748.1
109000.1
AUCA
108579.2
UUGG
1125
CGAA
UUUC
ACCA
GUGA
ACUA
UUUC
U
CCA
AD-
A-
CCCA
A-
AAAA
839-861
52749.1
108961.1
GCAA
108501.2
ACUU
CUCU
GAGA
CAAG
GUUG
UUUU
CUGG
U
GUC
AD-
A-
GACA
A-
AUUC
895-917
52750.1
108969.1
UUAA
108517.2
GAUG
UUCA
UUGA
ACAU
AUUA
CGAA
AUGU
U
CCA
AD-
A-
AACG
A-
UAUU
940-962
52751.1
108977.1
UGGG
108533.2
UGUA
AGAA
GUUC
CUAC
UCCC
AAAU
ACGU
A
UUC
AD-
A-
CUCC
A-
UUAG
1012-
52752.1
108985.1
AUAG
108549.2
AUUG
1034
UGAA
CUUC
GCAA
ACUA
UCUA
UGGA
A
GUA
AD-
A-
CAAC
A-
UAUU
1069-
52753.1
108993.1
AAAC
108565.2
CAAU
1091
AUUA
AUAA
UAUU
UGUU
GAAU
UGUU
A
GUC
AD-
A-
GAAA
A-
UAUA
1104-
52754.1
109001.1
UCAC
108581.2
GUUG
1126
GAAA
GUUU
CCAA
CGUG
CUAU
AUUU
A
CCC
AD-
A-
CUCU
A-
UAGA
847-869
52755.1
108962.1
CAAG
108503.2
CAUG
UUUU
AAAA
UCAU
ACUU
GUCU
GAGA
A
GUU
AD-
A-
ACAU
A-
UAUU
896-918
52756.1
108970.1
UAAU
108519.2
CGAU
UCAA
GUUG
CAUC
AAUU
GAAU
AAUG
A
UCC
AD-
A-
GGGA
A-
AACC
945-967
52757.1
108978.1
GAAC
108535.2
AUAU
UACA
UUGU
AAUA
AGUU
UGGU
CUCC
U
CAC
AD-
A-
UCCA
A-
AUUA
1013-
52758.1
108986.1
UAGU
108551.2
GAUU
1035
GAAG
GCUU
CAAU
CACU
CUAA
AUGG
U
AGU
AD-
A-
AACA
A-
AUAU
1070-
52759.1
108994.1
AACA
108567.2
UCAA
1092
UUAU
UAUA
AUUG
AUGU
AAUA
UUGU
U
UGU
AD-
A-
UGGC
A-
AUUG
1147-
52760.1
109002.1
AAUG
108583.2
CAUU
1169
UCCC
GGGG
CAAU
ACAU
GCAA
UGCC
U
AGU
AD-
A-
UCAG
A-
UAAU
881-
52761.1
108963.1
GUAG
108505.2
GUCC
903
UCCA
AUGG
UGGA
ACUA
CAUU
CCUG
A
AUA
AD-
A-
UUAA
A-
AUCU
899-921
52762.1
108971.1
UUCA
108521.2
AUUC
ACAU
GAUG
CGAA
UUGA
UAGA
AUUA
U
AUG
AD-
A-
GGAG
A-
AAAC
946-
52763.1
108979.1
AACU
108537.2
CAUA
968
ACAA
UUUG
AUAU
UAGU
GGUU
UCUC
U
CCA
AD-
A-
CCAU
A-
AAUU
1014-
52764.1
108987.1
AGUG
108553.2
AGAU
1036
AAGC
UGCU
AAUC
UCAC
UAAU
UAUG
U
GAG
AD-
A-
ACAA
A-
AAUA
1071-
52765.1
108995.1
ACAU
108569.2
UUCA
1093
UAUA
AUAU
UUGA
AAUG
AUAU
UUUG
U
UUG
AD-
A-
AAUG
A-
UUUG
1160-
52766.1
109003.1
CAAU
108585.2
UUUU
1182
CCCG
CCGG
GAAA
GAUU
ACAA
GCAU
A
UGG
AD-
A-
CAGG
A-
UUAA
882-904
52767.1
108964.1
UAGU
108507.2
UGUC
CCAU
CAUG
GGAC
GACU
AUUA
ACCU
A
GAU
AD-
A-
UUCA
A-
AUCC
903-925
52768.1
108972.1
ACAU
108523.2
AUCU
CGAA
AUUC
UAGA
GAUG
UGGA
UUGA
U
AUU
AD-
A-
GUUG
A-
UAUA
991-
52769.1
108980.1
GGCC
108539.2
UCUU
1013
UAGA
CUCU
GAAG
AGGC
AUAU
CCAA
A
CCA
AD-
A-
CAUA
A-
UAAU
1015-
52770.1
108988.1
GUGA
108555.2
UAGA
1037
AGCA
UUGC
AUCU
UUCA
AAUU
CUAU
A
GGA
AD-
A-
AACA
A-
AAGA
1074-
52771.1
108996.1
UUAU
108571.2
AUAU
1096
AUUG
UCAA
AAUA
UAUA
UUCU
AUGU
U
UUG
AD-
A-
GCAA
A-
AUCU
1163-
52772.1
109004.1
UCCC
108587.2
UUGU
1185
GGAA
UUUC
AACA
CGGG
AAGA
AUUG
U
CAU
AD-
A-
GGUA
A-
AAUU
884-906
52773.1
108965.1
GUCC
108509.2
AAUG
AUGG
UCCA
ACAU
UGGA
UAAU
CUAC
U
CUG
AD-
A-
AUCG
A-
UUUG
909-931
52774.1
108973.1
AAUA
108525.2
UGAU
GAUG
CCAU
GAUC
CUAU
ACAA
UCGA
A
UGU
AD-
A-
CCUA
A-
AUGG
997-
52775.1
108981.1
GAGA
108541.2
AGUA
1019
AGAU
UAUC
AUAC
UUCU
UCCA
CUAG
U
GCC
AD-
A-
GUUG
A-
UUGU
1051-
52776.1
108989.1
GAAG
108557.2
CUUU
1073
ACUG
CCAG
GAAA
UCUU
GACA
CCAA
A
CUC
AD-
A-
ACAU
A-
AAAG
1075-
52777.1
108997.1
UAUA
108573.2
AAUA
1097
UUGA
UUCA
AUAU
AUAU
UCUU
AAUG
U
UUU
AD-
A-
CAAU
A-
AAUC
1164-
52778.1
109005.1
CCCG
108589.2
UUUG
1186
GAAA
UUUU
ACAA
CCGG
AGAU
GAUU
U
GCA
AD-
A-
CUAC
A-
UUGC
1194-
52779.1
109013.1
UUGG
108605.2
UUUG
1216
GAUC
UGAU
ACAA
CCCA
AGCA
AGUA
A
GAA
AD-
A-
ACAA
A-
UAUA
1281-
52780.1
109021.1
CCUA
108621.2
UUUA
1303
AAUG
CCAU
GUAA
UUAG
AUAU
GUUG
A
UUU
AD-
A-
AUCC
A-
UUCU
1400-
52781.1
109029.1
AUCC
108637.2
GAAU
1422
AACA
CUGU
GAUU
UGGA
CAGA
UGGA
A
UCA
AD-
A-
AACU
A-
UCUU
1432-
52782.1
109037.1
GAGG
108653.2
UUAA
1454_
CAAA
AUUU
G21A
UUUA
GCCU
AAAG
CAGU
A
UCA
AD-
A-
AGAG
A-
ACAG
1913-
52783.1
109045.1
UAUG
108669.2
AUUU
1935
UGUA
UUAC
AAAA
ACAU
UCUG
ACUC
U
UGU
AD-
A-
AAUC
A-
AAAU
1165-
52784.1
109006.1
CCGG
108591.2
CU UUGU
1187
AAAA
UUUC
CAAA
CGGG
GAUU
AUUG
U
C
AD-
A-
UACU
A-
UUUG
1195-
52785.1
109014.1
UGGG
108607.2
CUUU
1217
AUCA
GUGA
CAAA
UCCC
GCAA
AAGU
A
AGA
AD-
A-
CAAC
A-
UUAU
1282-
52786.1
109022.1
CUAA
108623.2
AUUU
1304
AUGG
ACCA
UAAA
UUUA
UAUA
GGUU
A
GUU
AD-
A-
UUGA
A-
AAUU
1425-
52787.1
109030.1
AUGA
108639.2
UGCC
1447
ACUG
UCAG
AGGC
UUCA
AAAU
UUCA
U
AAG
AD-
A-
ACUG
A-
UCCU
1433-
52788.1
109038.1
AGGC
108655.2
UUUA
1455_
AAAU
AAUU
C21A
UUAA
UGCC
AAGG
UCAG
A
UUC
AD-
A-
GAGU
A-
UACA
1914-
52789.1
109046.1
AUGU
108671.2
GAUU
1936
GUAA
UUUA
AAAU
CACA
CUGU
UACU
A
CUG
AD-
A-
ACUU
A-
UUUU
1196-
52791.1
109015.1
GGGA
108609.2
GCUU
1218
UCAC
UGUG
AAAG
AUCC
CAAA
CAAG
A
UAG
AD-
A-
AUGG
A-
UUGG
1290-
52792.1
109023.1
UAAA
108625.2
UUUG
1312
UAUA
UUAU
ACAA
AUUU
ACCA
ACCA
A
UUU
AD-
A-
UGAA
A-
AAAU
1426-
52793.1
109031.1
UGAA
108641.2
UUGC
1448
CUGA
CUCA
GGCA
GUUC
AAUU
AUUC
U
AAA
AD-
A-
CUGA
A-
UGCC
1434-
52794.1
109039.1
GGCA
108657.2
UUUU
1456
AAUU
AAAU
UAAA
UUGC
AGGC
CUCA
A
GUU
AD-
A-
AGUA
A-
UUAC
1915-
52795.1
109047.1
UGUG
108673.2
AGAU
1937
UAAA
UUUU
AAUC
ACAC
UGUA
AUAC
A
UCU
AD-
A-
GAAA
A-
AAAC
1172-
52796.1
109008.1
ACAA
108595.2
ACCA
1194
AGAU
AAUC
UUGG
UUUG
UGUU
UUUU
U
CCG
AD-
A-
AGUG
A-
UUAG
1269-
52797.1
109016.1
UGGA
108611.2
GUUG
1291
GAAA
UUUU
ACAA
CUCC
CCUA
ACAC
A
UCA
AD-
A-
GUCU
A-
UAUA
1354-
52798.1
109024.1
CAAA
108627.2
ACCU
1376
AUGG
UCCA
AAGG
UUUU
UUAU
GAGA
A
CUU
AD-
A-
GAAU
A-
UAAA
1427-
52799.1
109032.1
GAAC
108643.2
UUUG
1449
UGAG
CCUC
GCAA
AGUU
AUUU
CAUU
A
CAA
AD-
A-
UGAG
A-
UUGC
1435-
52800.1
109040.1
GCAA
108659.2
CUUU
1457
AUUU
UAAA
AAAA
UUUG
GGCA
CCUC
A
AGU
AD-
A-
GUAU
A-
AUUA
1916-
52801.1
109048.1
GUGU
108675.2
CAGA
1938
AAAA
UUUU
AUCU
UACA
GUAA
CAUA
U
CUC
AD-
A-
AAAA
A-
AAAA
1173-
52802.1
109009.1
CAAA
108597.2
CACC
1195
GAUU
AAAU
UGGU
CUUU
GUUU
GUUU
U
UCC
AD-
A-
GUGU
A-
UUUA
1270-
52803.1
109017.1
GGAG
108613.2
GGUU
1292
AAAA
GUUU
CAAC
UCUC
CUAA
CACA
A
CUC
AD-
A-
AUGG
A-
UUAU
1362-
52804.1
109025.1
AAGG
108629.2
AGAG
1384
UUAU
UAUA
ACUC
ACCU
UAUA
UCCA
A
UUU
AD-
A-
AAUG
A-
UUAA
1428-
52805.1
109033.1
AACU
108645.2
AUUU
1450
GAGG
GCCU
CAAA
CAGU
UUUA
UCAU
A
UCA
AD-
A-
GAGG
A-
AUUG
1436-
52806.1
109041.1
CAAA
108661.2
CCUU
1458
UUUA
UUAA
AAAG
AUUU
GCAA
GCCU
U
CAG
AD-
A-
UAUG
A-
UAUU
1917-
52807.1
109049.1
UGUA
108677.2
ACAG
1939
AAAA
AUUU
UCUG
UUAC
UAAU
ACAU
A
ACU
AD-
A-
ACAA
A-
UAGA
1176-
52808.1
109010.1
AGAU
108599.2
AAAC
1198
UUGG
ACCA
UGUU
AAUC
UUCU
UUUG
A
UUU
AD-
A-
UGUG
A-
AUUU
1271-
52809.1
109018.1
GAGA
108615.2
AGGU
1293
AAAC
UGUU
AACC
UUCU
UAAA
CCAC
U
ACU
AD-
A-
UGGA
A-
UUUA
1363-
52810.1
109026.1
AGGU
108631.2
UAGA
1385
UAUA
GUAU
CUCU
AACC
AUAA
UUCC
A
AUU
AD-
A-
AUGA
A-
UUUA
1429-
52811.1
109034.1
ACUG
108647.2
AAUU
1451
AGGC
UGCC
AAAU
UCAG
UUAA
UUCA
A
UUC
AD-
A-
AGGC
A-
UAUU
1437-
52812.1
109042.1
AAAU
108663.2
GCCU
1459
UUAA
UUUA
AAGG
AAUU
CAAU
UGCC
A
UCA
AD-
A-
AAGA
A-
AAGU
1179-
52813.1
109011.1
UUUG
108601.2
AGAA
1201
GUGU
AACA
UUUC
CCAA
UACU
AUCU
U
UUG
AD-
A-
AAAC
A-
UAUU
1279-
52814.1
109019.1
AACC
108617.2
UACC
1301
UAAA
AUUU
UGGU
AGGU
AAAU
UGUU
A
UUC
AD-
A-
AUAC
A-
UUGG
1372-
52815.1
109027.1
UCUA
108633.2
UUGA
1394
UAAA
UUUU
AUCA
AUAG
ACCA
AGUA
A
UAA
AD-
A-
UGAA
A-
UUUU
1430-
52816.1
109035.1
CUGA
108649.2
AAAU
1452
GGCA
UUGC
AAUU
CUCA
UAAA
GUUC
A
AUU
AD-
A-
GGCA
A-
UUAU
1438-
52817.1
109043.1
AAUU
108665.2
UGCC
1460
UAAA
UUUU
AGGC
AAAU
AAUA
UUGC
A
CUC
AD-
A-
UUUU
A-
UUUG
1190-
52818.1
109012.1
CUAC
108603.2
UGAU
1212
UUGG
CCCA
GAUC
AGUA
ACAA
GAAA
A
ACA
AD-
A-
AACA
A-
AUAU
1280-
52819.1
109020.1
ACCU
108619.2
UUAC
1302
AAAU
CAUU
GGUA
UAGG
AAUA
UUGU
U
UUU
AD-
A-
UACU
A-
UUUG
1373-
52820.1
109028.1
CUAU
108635.2
GUUG
1395
AAAA
AUUU
UCAA
UAUA
CCAA
GAGU
A
AUA
AD-
A-
GAAC
A-
UUUU
1431-
52821.1
109036.1
UGAG
108651.2
UAAA
1453_
GCAA
UUUG
G21A
AUUU
CCUC
AAAA
AGUU
A
CAU
AD-
A-
CAGA
A-
AAGA
1912-
52822.1
109044.1
GUAU
108667.2
UUUU
1934_
GUGU
UACA
G21U
AAAA
CAUA
AUCU
CUCU
U
GUG
TABLE 10
Modified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences are the same as the sequences
listed in Table 8 except that they do not
contain GalNal conjugation.
Anti-
Sense
Sense
Oligo
Se-
Se-
quence
quence
(SEQ
(SEQ
ID
ID
NOS
NOS
1366-
1547-
1546,
1727,
respec-
respec-
tively,
tively,
in
in
order
Anti-
order
Sense
of
sense
of
Duplex
Oligo
appear-
Oligo
appear-
Name
Name
ance)
Name
ance)
AD-
A-
UfcA
A-
aAfa
52637.1
108817.1
fcAf
108307.2
GfaA
aUfu
fgGf
AfAf
aGfc
GfcU
uuAf
fcCf
aUfu
uUfc
GfuG
UfuU
fasA
f
fsc
AD-
A-
UfuA
A-
aAfa
52638.1
108825.1
fuUf
108323.2
UfaA
gUfu
fcUf
CfCf
aGfa
UfcU
ggAf
faGf
aCfa
uUfa
AfuA
UfuU
fasA
f
fsa
AD-
A-
GfcU
A-
uUfu
52639.1
108833.1
faUf
108339.2
UfaC
gUfu
faUf
AfGf
cGfu
AfcG
cuAf
faUf
aCfa
gUfa
UfaG
AfaA
fcsA
f
fsa
AD-
A-
GfgA
A-
aAfa
52640.1
108841.1
fcAf
108355.2
GfuC
uGfg
fuUf
UfCf
uAfa
UfuA
gaCf
faAf
cAfu
gAfc
GfuC
UfuU
fcsC
f
fsa
AD-
A-
CfaA
A-
aUfc
52641.1
108849.1
faAf
108371.2
AfaA
aCfu
fuAf
CfAf
uGfu
AfcA
ugAf
fuAf
gUfu
uUfu
UfuU
GfaU
fgsA
f
fsa
AD-
A-
AfcC
A-
uUfc
52642.1
108857.1
faGf
108387.2
UfuC
uGfa
fuUf
AfAf
uGfa
UfcA
uuUf
faAf
cAfc
gAfa
UfgG
GfaA
fusU
f
fsu
AD-
A-
CfaC
A-
aAfa
52643.1
108818.1
faAf
108309.2
AfgA
uUfa
faGf
AfGf
gAfg
CfuC
cuUf
fcUf
aAfu
uCfu
UfgU
UfuU
fgsA
f
fsa
AD-
A-
CfuA
A-
uUfu
52645.1
108834.1
fuGf
108341.2
UfuA
uUfa
fcAf
GfAf
uCfg
CfgA
ucUf
fuGf
aAfc
uAfa
AfuA
AfaA
fgsC
f
fsa
AD-
A-
UfcA
A-
aAfg
52647.1
108850.1
faCf
108373.2
AfcU
aUfa
fgAf
UfUf
uCfa
UfgA
aaUf
fuCf
aUfg
aGfu
UfuG
CfuU
fasG
f
fsu
AD-
A-
AfaC
A-
uAfu
52648.1
108858.1
fuGf
108389.2
AfuG
aGfa
fuAf
AfGf
gUfu
AfaC
cuUf
fuAf
cUfc
cAfu
AfgU
AfuA
fusC
f
fsc
AD-
A-
AfcA
A-
aAfa
52649.1
108819.1
faUf
108311.2
AfaG
uAfa
faAf
GfCf
gGfa
UfcC
gcUf
fuUf
uAfa
cUfu
UfuG
UfuU
fusG
f
fsa
AD-
A-
CfuC
A-
aUfc
52650.1
108827.1
fcAf
108327.2
UfuG
gAfg
faUf
CfCf
uUfu
AfaA
ggCf
faUf
uCfu
cAfa
GfgA
GfaU
fgsA
f
fsu
AD-
A-
CfgA
A-
uUfg
52651.1
108835.1
fuGf
108343.2
GfcU
uAfa
faAf
AfAf
aAfu
AfuU
uuUf
fuUf
uAfc
aGfc
AfuC
CfaA
fgsU
f
fsc
AD-
A-
GfuC
A-
uAfu
52652.1
108843.1
fuUf
108359.2
GfgA
aAfa
fcAf
GfAf
aAfg
CfuU
ucUf
fuGf
uUfa
uCfc
AfgA
AfuA
fcsC
f
fsa
AD-
A-
CfaA
A-
aAfa
52653.1
108851.1
fcAf
108375.2
GfaC
uAfu
fuGf
UfUf
aUfc
GfaU
aaAf
fcAf
uAfu
gUfc
GfuU
UfuU
fgsA
f
fsg
AD-
A-
AfcU
A-
uUfa
52654.1
108859.1
fgAf
108391.2
UfaU
gAfa
fgUf
GfAf
aGfu
AfcU
ucUf
faCf
uCfu
aUfa
CfaG
UfaA
fusU
f
fsc
AD-
A-
CfcA
A-
aAfa
52656.1
108828.1
fgAf
108329.2
UfcU
gCfc
fuGf
AfAf
aUfu
AfaU
uuGf
fcAf
gCfu
aGfa
CfuG
UfuU
fgsA
f
fsg
AD-
A-
GfaU
A-
aUfu
52657.1
108836.1
fgUf
108345.2
GfgC
aAfa
fuAf
AfAf
aAfa
UfuU
uuUf
fuAf
uUfa
gCfc
CfaU
AfaU
fcsG
f
fsu
AD-
A-
UfcU
A-
uUfa
52658.1
108844.1
fuAf
108361.2
UfgG
aAfg
faCf
AfCf
aAfa
UfuU
guCf
fgUf
uUfu
cCfa
AfaG
UfaA
fasC
f
fsc
AD-
A-
AfaC
A-
aAfa
52659.1
108852.1
faUf
108377.2
AfgA
aUfu
fcUf
UfGf
gAfu
AfuC
caAf
faGf
aUfa
uCfu
UfgU
UfuU
fusG
f
fsa
AD-
A-
CfuG
A-
uUfu
52660.1
108860.1
faGf
108393.2
AfuA
aAfg
fuGf
AfAf
uAfg
CfuA
uuCf
fcAf
uUfc
uAfu
UfcA
AfaA
fgsU
f
fsu
AD-
A-
AfaU
A-
aUfa
52661.1
108821.1
fuAf
108315.2
AfaA
aGfc
faGf
UfCf
aAfg
CfuU
gaGf
fcUf
cUfu
uUfu
AfaU
UfaU
fusG
f
fsu
AD-
A-
AfaA
A-
uAfa
52662.1
108829.1
fuCf
108331.2
CfaU
aAfg
faGf
AfUf
cAfa
UfuG
auCf
fcUf
uUfg
aUfg
AfuU
UfuA
fusU
f
fsg
AD-
A-
UfuC
A-
uAfa
52663.1
108837.1
faGf
108347.2
GfaC
uUfg
fcAf
GfGf
uGfu
AfcA
ccCf
fuGf
aAfc
gUfc
UfgA
UfuA
fasG
f
fsg
AD-
A-
GfgG
A-
aAfu
52664.1
108845.1
fcCf
108363.2
AfuG
aAfa
fuCf
UfUf
aUfu
AfaU
aaUf
fgAf
uUfg
cAfu
GfcC
AfuU
fcsU
f
fsu
AD-
A-
AfcA
A-
aAfa
52665.1
108853.1
fuAf
108379.2
AfaG
uUfu
faCf
GfAf
uGfa
UfcA
ucAf
fgUf
aAfu
cUfu
AfuG
UfuU
fusU
f
fsg
AD-
A-
AfgA
A-
uUfg
52666.1
108861.1
faCf
108395.2
UfaG
uAfc
fuUf
AfUf
uAfu
AfuA
auGf
faAf
uAfg
cUfa
UfuC
CfaA
fusU
f
fsc
AD-
A-
AfuU
A-
aAfu
52667.1
108822.1
faAf
108317.2
AfaA
gCfu
faAf
CfCf
gAfa
UfuC
ggAf
fuUf
gCfu
uUfu
UfaA
AfuU
fusU
f
fsg
AD-
A-
AfgA
A-
aUfc
52668.1
108830.1
fuUf
108333.2
GfuC
uGfc
fuAf
UfAf
aCfa
UfgU
uaGf
fuAf
cAfa
gAfc
AfuC
GfaU
fusU
f
fsg
AD-
A-
UfcA
A-
uUfa
52669.1
108838.1
fgUf
108349.2
AfgA
uGfg
fcCf
GfAf
aUfg
CfaU
ucCf
fgGf
cAfa
uCfu
CfuG
UfaA
fasA
f
fsg
AD-
A-
GfgC
A-
aAfa
52670.1
108846.1
fcAf
108365.2
UfaU
aAfu
fgUf
UfAf
cAfu
AfuG
uaAf
faCf
uUfu
aUfa
GfgC
UfuU
fcsC
f
fsu
AD-
A-
CfaU
A-
uAfa
52671.1
108854.1
faUf
108381.2
AfaA
uUfg
fgAf
AfUf
cUfg
CfaG
auCf
fuCf
aAfa
uUfu
UfaU
UfuA
fgsU
f
fsu
AD-
A-
UfaC
A-
uUfg
52672.1
108862.1
faUf
108397.2
AfcU
aUfa
fuGf
AfAf
uAfg
CfuA
uuUf
fcAf
aUfa
aGfu
UfgU
CfaA
fasG
f
fsu
AD-
A-
UfuU
A-
aUfa
52673.1
108823.1
fuAf
108319.2
AfcU
uUfg
faGf
UfUf
aGfg
CfcU
aaCf
fcUf
aAfu
aGfu
AfaA
UfaU
fasA
f
fsg
AD-
A-
UfuG
A-
uUfa
52674.1
108831.1
fcUf
108335.2
CfaU
aUfg
fcGf
UfUf
uCfu
AfgA
aaCf
fcGf
aUfa
aUfg
GfcA
UfaA
fasA
f
fsu
AD-
A-
CfaG
A-
uUfu
52675.1
108839.1
fuUf
108351.2
AfaG
gGfg
faCf
AfCf
cAfu
AfuG
guCf
fgUf
cCfa
cUfu
AfcU
AfaA
fgsA
f
fsa
AD-
A-
AfaA
A-
uUfu
52676.1
108847.1
fuUf
108367.2
GfaA
aAfu
faUf
GfAf
aUfg
CfaU
ucAf
faUf
uUfa
uUfc
AfuU
AfaA
fusG
f
fsg
AD-
A-
GfaU
A-
uAfg
52677.1
108855.1
fcAf
108383.2
AfuC
gUfc
faUf
UfUf
aAfa
UfuU
aaGf
faUf
aCfu
gAfu
GfaU
CfuA
fcsA
f
fsa
AD-
A-
AfcA
A-
uUfu
52678.1
108863.1
fuAf
108399.2
GfaC
uAfa
fuUf
AfCf
gUfa
UfaC
guUf
faAf
uAfu
gUfc
AfuG
AfaA
fusA
f
fsg
AD-
A-
UfuU
A-
aAfu
52679.1
108824.1
faUf
108321.2
AfaC
uGfu
fuAf
UfCf
gAfg
CfuC
gaAf
fuAf
cAfa
gUfu
UfaA
AfuU
fasA
f
fsa
AD-
A-
UfgC
A-
uUfu
52680.1
108832.1
fuAf
108337.2
AfcA
uGfu
fuCf
UfAf
gUfc
GfaC
uaAf
fgAf
cAfu
uGfu
AfgC
AfaA
fasA
f
fsa
AD-
A-
GfgG
A-
aAfg
52681.1
108840.1
faCf
108353.2
UfcU
aUfg
fuUf
GfUf
aAfg
CfuU
acCf
faAf
aUfg
aGfa
UfcC
CfuU
fcsA
f
fsa
AD-
A-
UfgA
A-
uUfg
52682.1
108848.1
fcAf
108369.2
AfgU
uAfu
fuUf
UfUf
uUfg
CfaA
aaAf
faAf
uAfu
aCfu
GfuC
CfaA
fasU
f
fsu
AD-
A-
AfuC
A-
aUfa
52683.1
108856.1
faGf
108385.2
GfaU
uCfu
fcAf
UfUf
uAfa
UfuA
aaAf
fuGf
gAfc
aUfc
UfgA
UfaU
fusC
f
fsa
AD-
A-
CfaU
A-
uUfu
52684.1
108864.1
faUf
108401.2
UfgA
aAfa
fcUf
CfUf
uGfu
AfcA
agUf
faGf
uUfa
uCfa
UfaU
AfaA
fgsU
f
fsa
AD-
A-
CfuU
A-
aAfg
52685.1
108872.1
fgAf
108417.2
UfuU
aCfu
fuGf
CfAf
aGfu
AfcU
ugAf
fcAf
gUfu
aAfa
CfaA
CfuU
fgsU
f
fsg
AD-
A-
CfuA
A-
uUfu
52686.1
108880.1
fcUf
108433.2
CfaC
uCfa
fuUf
AfCf
uUfu
AfaA
guUf
faAf
gAfa
gUfg
GfuA
AfaA
fgsA
f
fsa
AD-
A-
AfaG
A-
uUfa
52687.1
108888.1
faGf
108449.2
AfgU
cAfa
fuAf
CfUf
gUfu
AfaC
agUf
fuAf
uGfc
aCfu
UfcU
UfaA
fusC
f
fsu
AD-
A-
AfaA
A-
uUfu
52688.1
108896.1
fcAf
108465.2
GfaU
aGfa
fgCf
UfAf
uAfu
AfuA
uaUf
fgCf
cUfu
aUfc
GfuU
AfaA
fusU
f
fsu
AD-
A-
GfcA
A-
aUfu
52689.1
108904.1
fuAf
108481.2
UfcU
gUfc
fuUf
AfAf
uAfu
AfuA
uuGf
faAf
aCfu
aGfa
AfuG
AfaU
fcsU
f
fsg
AD-
A-
AfuA
A-
uUfu
52690.1
108865.1
fuAf
108403.2
UfuG
aAfc
faCf
UfAf
uUfg
CfaA
uaGf
fgUf
uUfu
cAfa
AfuA
AfaA
fusG
f
fsu
AD-
A-
GfaA
A-
uUfc
52691.1
108873.1
fcUf
108419.2
AfaG
cAfa
fuUf
CfUf
uUfg
CfaA
agUf
faAf
uGfa
cUfu
GfuU
GfaA
fcsA
f
fsa
AD-
A-
UfaC
A-
aUfu
52692.1
108881.1
fuUf
108435.2
UfcA
cAfa
fcUf
CfAf
uUfu
AfaA
ugUf
faGf
uGfa
uGfa
AfgU
AfaU
fasG
f
fsa
AD-
A-
AfgA
A-
aUfu
52693.1
108889.1
fgCf
108451.2
AfaG
aAfc
fuUf
UfAf
aGfu
AfcU
uaGf
faAf
uUfg
cUfu
CfuC
AfaU
fusU
f
fsc
AD-
A-
GfaU
A-
aAfg
52694.1
108897.1
faAf
108467.2
GfuC
uAfg
fuUf
CfAf
uGfa
UfcA
ugCf
faAf
uAfu
gAfc
UfaU
CfuU
fcsU
f
fsu
AD-
A-
CfaU
A-
uAfu
52695.1
108905.1
faGf
108483.2
UfuC
uCfa
fuUf
AfAf
uUfa
UfaA
uuUf
faAf
gAfc
gAfa
UfaU
AfuA
fgsC
f
fsu
AD-
A-
UfaU
A-
aUfu
52696.1
108866.1
faAf
108405.2
UfuU
aCfu
fgAf
AfCf
cUfu
AfaG
guAf
fuCf
gUfu
aAfa
UfaU
AfaU
fasU
f
fsg
AD-
A-
AfaC
A-
uUfu
52697.1
108874.1
fuCf
108421.2
CfaA
aAfc
fgUf
UfCf
uUfu
AfaA
gaGf
faCf
uUfg
uUfg
AfgU
AfaA
fusC
f
fsa
AD-
A-
AfcU
A-
uAfu
52698.1
108882.1
fuCf
108437.2
UfuC
aAfc
faCf
AfAf
uUfu
AfaA
uuGf
fgUf
uUfg
gAfa
AfaG
AfuA
fusA
f
fsg
AD-
A-
GfaG
A-
aAfu
52699.1
108890.1
fcAf
108453.2
UfaA
aCfu
fgUf
AfAf
uAfg
CfuA
uuAf
faCf
gUfu
uUfa
GfcU
AfuU
fcsU
f
fsu
AD-
A-
AfaC
A-
uAfu
52700.1
108898.1
fcAf
108469.2
UfuG
aCfa
faCf
GfCf
uAfu
AfuA
gcUf
fgUf
gUfu
cAfa
GfgU
AfuA
fusU
f
fsa
AD-
A-
AfgU
A-
uUfc
52701.1
108906.1
fcAf
108485.2
UfaU
aAfu
fuUf
AfAf
cUfu
AfaG
uuAf
faAf
uUfu
aUfa
GfaC
GfaA
fusA
f
fsu
AD-
A-
AfgU
A-
uUfu
52702.1
108867.1
fcAf
108407.2
AfcC
aAfa
fuCf
AfUf
uUfc
GfaA
auUf
fgAf
uUfu
gGfu
GfaC
AfaA
fusU
f
fsg
AD-
A-
CfuU
A-
uUfc
52703.1
108875.1
fgAf
108423.2
UfuC
aAfg
fuAf
CfCf
gGfa
UfcC
ggCf
fuAf
uUfu
gAfa
CfaA
GfaA
fgsU
f
fsu
AD-
A-
CfuU
A-
aUfa
52704.1
108883.1
fcAf
108439.2
UfuU
aCfa
fcAf
AfAf
cUfu
AfaG
uuUf
fuGf
gUfu
aAfa
GfaA
UfaU
fgsU
f
fsa
AD-
A-
CfaA
A-
uUfg
52705.1
108891.1
fcUf
108455.2
AfaU
aAfc
fuAf
UfAf
aGfu
AfcU
uaGf
fuAf
uUfa
aUfu
GfuU
CfaA
fgsC
f
fsu
AD-
A-
AfcC
A-
uUfa
52706.1
108899.1
faAf
108471.2
UfuU
cAfg
fgAf
CfAf
cUfa
UfaG
ugCf
fuCf
uGfu
aAfa
UfgG
UfaA
fusU
f
fsu
AD-
A-
GfaA
A-
uAfg
52707.1
108907.1
fcCf
108487.2
AfgA
cAfc
faAf
AfGf
uUfu
AfaA
cuGf
fuUf
uGfg
uCfu
GfuU
CfuA
fcsU
f
fsu
AD-
A-
GfaA
A-
uUfg
52708.1
108868.1
fuAf
108409.2
AfgU
uGfu
fuCf
CfAf
aAfg
CfuU
ugAf
fgAf
cAfu
aCfu
AfuU
CfaA
fcsU
f
fsu
AD-
A-
UfgA
A-
uUfu
52709.1
108876.1
faAf
108425.2
UfcU
gCfc
fuCf
UfCf
uAfg
CfuA
gaGf
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cCfa
UfgG
uuUf
fuAf
aGfg
aAfu
UfuG
AfuA
fusU
f
fsu
AD-
A-
AfuC
A-
uUfc
52781.1
109029.1
fcAf
108637.2
UfgA
uCfc
faUf
AfAf
cUfg
CfaG
uuGf
faUf
gAfu
uCfa
GfgA
GfaA
fusC
f
fsa
AD-
A-
AfaC
A-
uCfu
52782.1
109037.1
fuGf
108653.2
UfuU
aGfg
faAf
CfAf
aUfu
AfaU
ugCf
fuUf
cUfc
aAfa
AfgU
AfgA
fusC
f
fsa
AD-
A-
AfgA
A-
aCfa
52783.1
109045.1
fgUf
108669.2
GfaU
aUfg
fuUf
UfGf
uUfa
UfaA
caCf
faAf
aUfa
aUfc
CfuC
UfgU
fusG
f
fsu
AD-
A-
AfaU
A-
aAfa
52784.1
109006.1
fcCf
108591.2
UfcU
cGfg
fuUf
AfAf
gUfu
AfaC
uuCf
faAf
cGfg
aGfa
GfaU
UfuU
fusG
f
fsc
AD-
A-
UfaC
A-
uUfu
52785.1
109014.1
fuUf
108607.2
GfcU
gGfg
fuUf
AfUf
gUfg
CfaC
auCf
faAf
cCfa
aGfc
AfgU
AfaA
fasG
f
fsa
AD-
A-
CfaA
A-
uUfa
52786.1
109022.1
fcCf
108623.2
UfaU
uAfa
fuUf
AfUf
aCfc
GfgU
auUf
faAf
uAfg
aUfa
GfuU
UfaA
fgsU
f
fsu
AD-
A-
UfuG
A-
aAfu
52787.1
109030.1
faAf
108639.2
UfuG
uGfa
fcCf
AfCf
uCfa
UfgA
guUf
fgGf
cAfu
cAfa
UfcA
AfuU
fasA
f
fsg
AD-
A-
AfcU
A-
uCfc
52788.1
109038.1
fgAf
108655.2
UfuU
gGfc
fuAf
AfAf
aAfu
AfuU
uuGf
fuAf
cCfu
aAfa
CfaG
GfgA
fusU
f
fsc
AD-
A-
GfaG
A-
uAfc
52789.1
109046.1
fuAf
108671.2
AfgA
uGfu
fuUf
GfUf
uUfu
AfaA
acAf
faAf
cAfu
uCfu
AfcU
GfuA
fcsU
f
fsg
AD-
A-
AfcU
A-
uUfu
52791.1
109015.1
fuGf
108609.2
UfgC
gGfa
fuUf
UfCf
uGfu
AfcA
gaUf
faAf
cCfc
gCfa
AfaG
AfaA
fusA
f
fsg
AD-
A-
AfuG
A-
uUfg
52792.1
109023.1
fgUf
108625.2
GfuU
aAfa
fuGf
UfAf
uUfa
UfaA
uaUf
fcAf
uUfa
aAfc
CfcA
CfaA
fusU
f
fsu
AD-
A-
UfgA
A-
aAfa
52793.1
109031.1
faUf
108641.2
UfuU
gAfa
fgCf
CfUf
cUfc
GfaG
agUf
fgCf
uCfa
aAfa
UfuC
UfuU
fasA
f
fsa
AD-
A-
CfuG
A-
uGfc
52794.1
109039.1
faGf
108657.2
CfuU
gCfa
fuUf
AfAf
aAfa
UfuU
uuUf
faAf
gCfc
aAfg
UfcA
GfcA
fgsU
f
fsu
AD-
A-
AfgU
A-
uUfa
52795.1
109047.1
faUf
108673.2
CfaG
gUfg
faUf
UfAf
uUfu
AfaA
uaCf
faUf
aCfa
cUfg
UfaC
UfaA
fusC
f
fsu
AD-
A-
GfaA
A-
aAfa
52796.1
109008.1
faAf
108595.2
CfaC
cAfa
fcAf
AfGf
aAfu
AfuU
cuUf
fuGf
uGfu
gUfg
UfuU
UfuU
fcsC
f
fsg
AD-
A-
AfgU
A-
uUfa
52797.1
109016.1
fgUf
108611.2
GfgU
gGfa
fuGf
GfAf
uUfu
AfaA
ucUf
fcAf
cCfa
aCfc
CfaC
UfaA
fusC
f
fsa
AD-
A-
GfuC
A-
uAfu
52798.1
109024.1
fuCf
108627.2
AfaC
aAfa
fcUf
AfUf
uCfc
GfgA
auUf
faGf
uUfg
gUfu
AfgA
AfuA
fcsU
f
fsu
AD-
A-
GfaA
A-
uAfa
52799.1
109032.1
fuGf
108643.2
AfuU
aAfc
fuGf
UfGf
cCfu
AfgG
caGf
fcAf
uUfc
aAfu
AfuU
UfuA
fcsA
f
fsa
AD-
A-
UfgA
A-
uUfg
52800.1
109040.1
fgGf
108659.2
CfcU
cAfa
fuUf
AfUf
uAfa
UfuA
auUf
faAf
uGfc
aGfg
CfuC
CfaA
fasG
f
fsu
AD-
A-
GfuA
A-
aUfu
52801.1
109048.1
fuGf
108675.2
AfcA
uGfu
fgAf
AfAf
uUfu
AfaA
uuAf
fuCf
cAfc
uGfu
AfuA
AfaU
fcsU
f
fsc
AD-
A-
AfaA
A-
aAfa
52802.1
109009.1
faCf
108597.2
AfcA
aAfa
fcCf
GfAf
aAfa
UfuU
ucUf
fgGf
uUfg
uGfu
UfuU
UfuU
fusC
f
fsc
AD-
A-
GfuG
A-
uUfu
52803.1
109017.1
fuGf
108613.2
AfgG
gAfg
fuUf
AfAf
gUfu
AfaC
uuCf
faAf
uCfc
cCfu
AfcA
AfaA
fcsU
f
fsc
AD-
A-
AfuG
A-
uUfa
52804.1
109025.1
fgAf
108629.2
UfaG
aGfg
faGf
UfUf
uAfu
AfuA
aaCf
fcUf
cUfu
cUfa
CfcA
UfaA
fusU
f
fsu
AD-
A-
AfaU
A-
uUfa
52805.1
109033.1
fgAf
108645.2
AfaU
aCfu
fuUf
GfAf
gCfc
GfgC
ucAf
faAf
gUfu
aUfu
CfaU
UfaA
fusC
f
fsa
AD-
A-
GfaG
A-
aUfu
52806.1
109041.1
fgCf
108661.2
GfcC
aAfa
fuUf
UfUf
uUfa
UfaA
aaUf
faAf
uUfg
gGfc
CfcU
AfaU
fcsA
f
fsg
AD-
A-
UfaU
A-
uAfu
52807.1
109049.1
fgUf
108677.2
UfaC
gUfa
faGf
AfAf
aUfu
AfaU
uuUf
fcUf
aCfa
gUfa
CfaU
AfuA
fasC
f
fsu
AD-
A-
AfcA
A-
uAfg
52808.1
109010.1
faAf
108599.2
AfaA
gAfu
faCf
UfUf
aCfc
GfgU
aaAf
fgUf
uCfu
uUfu
UfuG
CfuA
fusU
f
fsu
AD-
A-
UfgU
A-
aUfu
52809.1
109018.1
fgGf
108615.2
UfaG
aGfa
fgUf
AfAf
uGfu
AfcA
uuUf
faCf
cUfc
cUfa
CfaC
AfaU
fasC
f
fsu
AD-
A-
UfgG
A-
uUfu
52810.1
109026.1
faAf
108631.2
AfuA
gGfu
fgAf
UfAf
gUfa
UfaC
uaAf
fuCf
cCfu
uAfu
UfcC
AfaA
fasU
f
fsu
AD-
A-
AfuG
A-
uUfu
52811.1
109034.1
faAf
108647.2
AfaA
cUfg
fuUf
AfGf
uGfc
GfcA
cuCf
faAf
aGfu
uUfu
UfcA
AfaA
fusU
f
fsc
AD-
A-
AfgG
A-
uAfu
52812.1
109042.1
fcAf
108663.2
UfgC
aAfu
fcUf
UfUf
uUfu
AfaA
aaAf
faGf
uUfu
gCfa
GfcC
AfuA
fusC
f
fsa
AD-
A-
AfaG
A-
aAfg
52813.1
109011.1
faUf
108601.2
UfaG
uUfg
faAf
GfUf
aAfc
GfuU
acCf
fuUf
aAfa
cUfa
UfcU
CfuU
fusU
f
fsg
AD-
A-
AfaA
A-
uAfu
52814.1
109019.1
fcAf
108617.2
UfuA
aCfc
fcCf
UfAf
aUfu
AfaU
uaGf
fgGf
gUfu
uAfa
GfuU
AfuA
fusU
f
fsc
AD-
A-
AfuA
A-
uUfg
52815.1
109027.1
fcUf
108633.2
GfuU
cUfa
fgAf
UfAf
uUfu
AfaA
uaUf
fuCf
aGfa
aAfc
GfuA
CfaA
fusA
f
fsa
AD-
A-
UfgA
A-
uUfu
52816.1
109035.1
faCf
108649.2
UfaA
uGfa
faUf
GfGf
uUfg
CfaA
ccUf
faUf
cAfg
uUfa
UfuC
AfaA
fasU
f
fsu
AD-
A-
GfgC
A-
uUfa
52817.1
109043.1
faAf
108665.2
UfuG
aUfu
fcCf
UfAf
uUfu
AfaA
uaAf
fgGf
aUfu
cAfa
UfgC
UfaA
fcsU
f
fsc
AD-
A-
UfuU
A-
uUfu
52818.1
109012.1
fuCf
108603.2
GfuG
uAfc
faUf
UfUf
cCfc
GfgG
aaGf
faUf
uAfg
cAfc
AfaA
AfaA
fasC
f
fsa
AD-
A-
AfaC
A-
aUfa
52819.1
109020.1
faAf
108619.2
UfuU
cCfu
faCf
AfAf
cAfu
AfuG
uuAf
fgUf
gGfu
aAfa
UfgU
UfaU
fusU
f
fsu
AD-
A-
UfaC
A-
uUfu
52820.1
109028.1
fuCf
108635.2
GfgU
uAfu
fuGf
AfAf
aUfu
AfaU
uuAf
fcAf
uAfg
aCfc
AfgU
AfaA
fasU
f
fsa
AD-
A-
GfaA
A-
uUfu
52821.1
109036.1
fcUf
108651.2
UfuA
gAfg
faAf
GfCf
uUfu
AfaA
gcCf
fuUf
uCfa
uAfa
GfuU
AfaA
fcsA
f
fsu
AD-
A-
CfaG
A-
aAfg
52822.1
109044.1
faGf
108667.2
AfuU
uAfu
fuUf
GfUf
uAfc
GfuA
acAf
faAf
uAfc
aAfu
UfcU
CfuU
fgsU
f
fsg
TABLE 11
Results of single dose screen using ANGPTL3 GalNac-conjugated dsRNA
Modified siRNAs were tested by transfection in Hep3b cells and by free-
uptake in primary cynomolgus monkey (PCH) cells at the above-stated doses.
500 nM
100 nM
10 nM
PCH
PCH
PCH
STDEV
STDEV
STDEV
STDEV
STDEV
10 nM
0.1 nM
Celsis
Celsis
Celsis
10 nM
0.1 nM
500 nM
100 nM
10 nM
DUPLEX ID
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
AD1955/naïve FU
0.93
0.93
1.01
0.91
1.17
0.02
0.08
0.09
0.00
0.07
AD1955/naïve FU
1.02
1.09
1.07
1.07
0.92
0.06
0.04
0.02
0.00
0.03
AD1955/naïve FU
1.06
0.99
0.93
1.02
0.93
0.03
0.00
0.09
0.01
0.02
AD1955/naïve FU
1.05
0.90
1.05
1.03
1.03
0.04
0.02
0.01
0.05
0.01
AD1955/naïve FU
1.06
1.08
0.90
0.97
1.03
0.02
0.01
0.02
0.04
0.09
AD1955/naïve FU
0.90
1.03
1.05
1.00
0.94
0.04
0.03
0.01
0.04
0.05
AD-45165 (TTR)
0.91
0.98
1.06
0.98
0.96
0.05
0.01
0.05
0.00
0.00
AD-52953.1
0.06
0.34
0.15
0.17
0.46
0.00
0.01
0.00
0.01
0.01
AD-52954.1
0.09
0.39
0.17
0.20
0.55
0.00
0.01
0.00
0.01
0.00
AD-52955.1
0.11
0.59
0.38
0.41
0.75
0.01
0.04
0.02
0.01
0.12
AD-52956.1
0.31
0.94
0.79
0.94
1.17
0.01
0.00
0.02
0.06
0.02
AD-52957.1
0.13
0.61
0.35
0.38
0.73
0.01
0.00
0.01
0.00
0.04
AD-52958.1
0.19
0.74
0.66
0.71
0.97
0.01
0.01
0.02
0.07
0.06
AD-52960.1
0.14
0.59
0.31
0.32
0.55
0.01
0.01
0.00
0.02
0.02
AD-52961.1
0.05
0.66
0.27
0.24
0.49
0.00
0.00
0.00
0.02
0.02
AD-52962.1
0.83
0.89
1.03
1.02
1.26
0.02
0.05
0.07
0.07
0.07
AD-52963.1
0.07
0.72
0.46
0.56
0.91
0.00
0.00
0.00
0.00
0.06
AD-52964.1
0.13
0.73
0.41
0.47
0.68
0.01
0.03
0.02
0.03
0.01
AD-52965.1
0.07
0.44
0.16
0.18
0.43
0.00
0.01
0.00
0.01
0.01
AD-52966.1
0.12
0.76
0.67
0.72
0.96
0.00
0.02
0.05
0.01
0.01
AD-52967.1
0.10
0.75
0.44
0.58
0.89
0.01
0.04
0.02
0.03
0.04
AD-52968.1
1.01
0.96
0.87
0.91
1.15
0.00
0.01
0.09
0.03
0.02
AD-52969.1
0.04
0.46
0.22
0.29
0.59
0.00
0.00
0.01
0.02
0.04
AD-52970.1
0.06
0.45
0.27
0.30
0.51
0.00
0.00
0.01
0.02
0.00
AD-52971.1
0.08
0.55
0.20
0.22
0.45
0.00
0.00
0.01
0.02
0.05
AD-52972.1
0.10
0.73
0.41
0.49
0.81
0.00
0.01
0.01
0.02
0.01
AD-52973.1
0.11
0.73
0.36
0.46
0.75
0.01
0.01
0.03
0.02
0.02
AD-52974.1
1.00
0.95
1.00
1.09
1.27
0.01
0.01
0.08
0.05
0.06
AD-52975.1
0.07
0.54
0.25
0.34
0.66
0.00
0.01
0.01
0.01
0.03
AD-52976.1
0.17
0.59
0.35
0.41
0.65
0.00
0.02
0.04
0.01
0.01
AD-52977.1
0.07
0.45
0.16
0.25
0.50
0.01
0.02
0.00
0.02
0.03
AD-52978.1
0.10
0.72
0.39
0.53
0.77
0.00
0.02
0.00
0.08
0.03
AD-52979.1
0.54
0.92
0.99
1.12
1.28
0.01
0.02
0.02
0.04
0.05
AD-52980.1
0.29
0.85
0.67
0.85
1.03
0.01
0.01
0.05
0.05
0.04
AD-52981.1
0.07
0.44
0.20
0.26
0.59
0.01
0.02
0.00
0.00
0.03
AD-52982.1
0.28
0.87
0.67
0.99
1.14
0.01
0.01
0.04
0.00
0.01
AD-52983.1
0.06
0.40
0.14
0.40
0.46
0.00
0.00
0.01
0.05
0.02
AD-52984.1
0.29
0.87
0.66
0.74
1.09
0.01
0.02
0.01
0.00
0.00
AD-52985.1
0.72
0.87
0.89
1.18
1.22
0.03
0.00
0.05
0.03
0.16
AD-52986.1
0.08
0.47
0.24
0.30
0.48
0.00
0.02
0.02
0.00
0.06
AD-52987.1
0.16
0.83
0.42
0.73
1.09
0.00
0.00
0.01
0.02
0.02
AD-52988.1
0.11
0.73
0.42
0.60
0.96
0.01
0.04
0.00
0.00
0.10
AD-52989.1
0.05
0.48
0.15
0.42
0.46
0.00
0.02
0.00
0.02
0.00
AD-52990.1
0.14
0.86
0.33
0.45
0.77
0.00
0.01
0.00
0.02
0.05
AD-52991.1
0.16
0.86
0.58
0.69
1.05
0.00
0.00
0.02
0.00
0.02
AD-52992.1
0.08
0.65
0.42
0.56
0.90
0.00
0.01
0.02
0.01
0.00
AD-52993.1
0.13
0.87
0.53
0.76
1.08
0.02
0.03
0.04
0.04
0.00
AD-52994.1
0.10
0.52
0.28
0.33
0.53
0.01
0.00
0.02
0.00
0.01
AD-52995.1
0.06
0.56
0.19
0.41
0.60
0.00
0.01
0.04
0.02
0.05
AD-52996.1
0.09
0.68
0.26
0.47
0.68
0.00
0.03
0.01
0.04
0.01
AD-52997.1
0.59
1.03
0.87
0.51
1.25
0.05
0.01
0.00
0.01
0.01
AD-52998.1
0.09
0.79
0.44
0.55
0.85
0.00
0.00
0.04
0.03
0.10
AD-52999.1
0.08
0.57
0.17
0.36
0.84
0.01
0.00
0.01
0.02
0.00
AD-53000.1
0.38
0.94
0.58
0.67
0.85
0.01
0.02
0.03
0.03
0.02
AD-53001.1
0.05
0.48
0.21
0.18
0.40
0.00
0.00
0.01
0.00
0.05
AD-53002.1
0.07
0.65
0.43
0.48
0.80
0.00
0.05
0.04
0.01
0.02
AD-53003.1
0.05
0.46
0.31
0.34
0.56
0.01
0.01
0.00
0.02
0.05
AD-53004.1
0.05
0.36
0.29
0.66
0.57
0.00
0.01
0.03
0.35
0.02
AD-53005.1
0.05
0.72
0.32
0.58
0.83
0.01
0.00
0.01
0.29
0.00
AD-53006.1
0.21
0.82
0.66
0.77
1.03
0.01
0.00
0.02
0.07
0.02
AD-53007.1
0.12
0.76
0.55
0.73
0.74
0.01
0.00
0.00
0.08
0.20
AD-53008.1
0.07
0.68
0.28
0.36
0.84
0.00
0.02
0.01
0.05
0.03
AD-53009.1
0.10
0.61
0.48
0.60
0.91
0.00
0.02
0.01
0.01
0.06
AD-53010.1
0.05
0.58
0.47
0.54
0.84
0.00
0.02
0.00
0.02
0.03
AD-53011.1
0.07
0.65
0.29
0.34
0.84
0.00
0.03
0.07
0.01
0.04
AD-53012.1
0.06
0.55
0.36
0.45
0.70
0.00
0.03
0.02
0.02
0.00
AD-53013.1
0.11
0.85
0.59
0.70
1.01
0.00
0.00
0.03
0.03
0.02
AD-53014.1
0.16
0.78
0.61
0.78
1.11
0.00
0.02
0.01
0.05
0.00
AD-53015.1
0.03
0.35
0.25
0.37
0.46
0.01
0.01
0.01
0.00
0.01
AD-53016.1
0.03
0.56
0.40
0.58
1.01
0.00
0.01
0.02
0.06
0.09
AD-53017.1
0.07
0.71
0.64
0.78
0.98
0.00
0.01
0.01
0.05
0.00
AD-53018.1
0.30
0.96
0.75
0.97
1.14
0.00
0.02
0.02
0.03
0.05
AD-53019.1
0.27
0.99
0.77
1.05
1.31
0.00
0.01
0.01
0.04
0.00
AD-53020.1
0.04
0.64
0.32
0.45
0.69
0.00
0.00
0.03
0.02
0.03
AD-53021.1
0.04
0.68
0.36
0.48
0.70
0.01
0.01
0.02
0.07
0.00
AD-53022.1
0.05
0.76
0.36
0.59
1.04
0.01
0.01
0.02
0.03
0.06
AD-53023.1
0.10
0.83
0.69
0.84
0.97
0.01
0.01
0.06
0.02
0.01
AD-53024.1
0.09
0.44
0.23
0.23
0.44
0.00
0.00
0.03
0.01
0.02
AD-53025.1
0.09
0.87
0.58
0.80
1.09
0.00
0.03
0.01
0.04
0.04
AD-53026.1
0.05
0.60
0.35
0.46
0.77
0.01
0.01
0.02
0.05
0.03
AD-53027.1
0.02
0.32
0.26
0.30
0.45
0.00
0.01
0.02
0.03
0.02
AD-53028.1
0.19
0.82
0.77
0.95
1.04
0.01
0.04
0.05
0.01
0.03
AD-53029.1
0.02
0.52
0.32
0.41
0.72
0.00
0.00
0.01
0.02
0.07
AD-53030.1
0.09
0.42
0.15
0.16
0.46
0.00
0.00
0.00
0.00
0.02
AD-53031.1
0.12
0.79
0.63
0.73
1.04
0.02
0.05
0.02
0.04
0.03
AD-53032.1
0.12
0.71
0.41
0.59
0.90
0.01
0.00
0.02
0.04
0.00
AD-53033.1
0.02
0.48
0.20
0.21
0.51
0.00
0.02
0.02
0.01
0.00
AD-53034.1
0.04
0.52
0.31
0.36
0.71
0.00
0.01
0.07
0.02
0.01
AD-53035.1
0.02
0.63
0.34
0.50
0.85
0.00
0.02
0.03
0.00
0.03
AD-53036.1
0.10
0.57
0.31
0.35
0.65
0.01
0.01
0.03
0.03
0.01
AD-53037.1
0.08
0.47
0.27
0.36
0.60
0.00
0.02
0.01
0.03
0.01
AD-53038.1
0.05
0.85
0.48
0.63
1.08
0.00
0.05
0.00
0.02
0.05
AD-53039.1
0.08
0.82
0.45
0.64
0.97
0.00
0.01
0.01
0.03
0.00
AD-53040.1
0.05
0.79
0.46
0.62
0.97
0.01
0.01
0.01
0.05
0.06
AD-53041.1
0.06
0.72
0.59
0.61
0.86
0.00
0.01
0.05
0.06
0.03
AD-53042.1
0.08
0.85
0.30
0.35
0.81
0.01
0.00
0.00
0.03
0.03
AD-53043.1
0.63
1.00
0.92
1.04
1.07
0.03
0.00
0.06
0.03
0.07
AD-53044.1
0.05
0.91
0.35
0.61
0.97
0.01
0.01
0.01
0.04
0.02
AD-53045.1
0.20
1.00
0.85
1.00
0.98
0.00
0.03
0.04
0.01
0.04
AD-53046.1
0.07
0.70
0.44
0.62
1.12
0.00
0.01
0.03
0.00
0.09
AD-53059.1
0.35
1.04
0.75
0.85
0.86
0.01
0.01
0.03
0.02
0.04
AD-53060.1
0.34
0.85
0.72
0.96
0.82
0.00
0.01
0.02
0.01
0.02
AD-53061.1
0.17
0.94
0.36
0.37
0.59
0.00
0.00
0.02
0.00
0.02
AD-53062.1
0.09
0.76
0.43
0.47
0.69
0.01
0.01
0.01
0.03
0.01
AD-53063.1
0.06
0.48
0.18
0.16
0.25
0.00
0.01
0.01
0.01
0.02
AD-53064.1
0.07
0.59
0.22
0.22
0.48
0.01
0.02
0.01
0.02
0.06
AD-53065.1
0.08
0.97
0.45
0.39
0.64
0.01
0.01
0.02
0.01
0.01
AD-53066.1
0.12
0.99
0.73
0.67
0.88
0.01
0.03
0.01
0.01
0.05
AD-53067.1
0.12
1.08
0.59
0.60
0.79
0.00
0.12
0.01
0.01
0.03
AD-53068.1
0.09
0.98
0.46
0.59
0.83
0.00
0.03
0.04
0.07
0.05
AD-53069.1
0.04
0.69
0.35
0.43
0.59
0.00
0.01
0.01
0.04
0.01
AD-53070.1
0.17
1.12
0.88
0.83
0.98
0.00
0.01
0.04
0.00
0.01
AD-53071.1
0.07
0.70
0.23
0.23
0.43
0.00
0.00
0.02
0.00
0.01
AD-53072.1
0.10
0.90
0.49
0.48
0.75
0.01
0.05
0.00
0.01
0.02
AD-53073.1
0.07
0.63
0.27
0.30
0.43
0.00
0.00
0.01
0.01
0.00
AD-53074.1
0.07
0.88
0.46
0.49
0.62
0.01
0.08
0.01
0.06
0.03
AD-53075.1
0.05
0.76
0.29
0.35
0.50
0.01
0.01
0.00
0.02
0.03
AD-53076.1
0.09
0.80
0.31
0.40
0.54
0.01
0.01
0.02
0.05
0.02
AD-53077.1
0.07
0.96
0.29
0.28
0.49
0.00
0.03
0.00
0.01
0.01
AD-53078.1
0.16
0.95
0.51
0.51
0.70
0.00
0.04
0.01
0.01
0.06
AD-53079.1
0.08
0.96
0.59
0.67
0.83
0.00
0.02
0.01
0.03
0.01
AD-53080.1
0.04
0.63
0.20
0.22
0.43
0.00
0.01
0.00
0.01
0.01
AD-53081.1
0.16
1.02
0.63
0.75
0.87
0.00
0.09
0.00
0.02
0.05
AD-53082.1
0.06
0.94
0.50
0.52
0.66
0.01
0.06
0.02
0.03
0.03
AD-53083.1
0.14
0.87
0.48
0.50
0.80
0.01
0.02
0.04
0.06
0.01
AD-53084.1
0.12
0.95
0.50
0.47
0.72
0.01
0.03
0.04
0.00
0.00
AD-53085.1
0.27
1.02
0.68
0.81
0.99
0.01
0.01
0.01
0.05
0.02
AD-53086.1
0.05
0.60
0.26
0.25
0.48
0.00
0.01
0.03
0.00
0.01
AD-53087.1
0.05
0.56
0.32
0.39
0.53
0.00
0.01
0.01
0.03
0.02
AD-53088.1
0.09
0.89
0.53
0.69
0.87
0.00
0.01
0.02
0.04
0.02
AD-53089.1
0.29
0.97
0.58
0.57
0.78
0.01
0.00
0.02
0.02
0.02
AD-53090.1
0.13
0.86
0.56
0.55
0.73
0.00
0.01
0.01
0.03
0.00
AD-53091.1
0.12
0.82
0.27
0.35
0.66
0.00
0.03
0.03
0.01
0.07
AD-53092.1
0.05
0.66
0.26
0.29
0.42
0.00
0.01
0.02
0.04
0.02
AD-53093.1
0.08
0.68
0.36
0.44
0.55
0.00
0.02
0.03
0.04
0.10
AD-53094.1
0.32
1.00
1.05
0.92
1.11
0.02
0.01
0.01
0.00
0.03
AD-53095.1
0.14
0.77
0.29
0.29
0.49
0.00
0.02
0.00
0.01
0.01
AD-53096.1
0.30
0.96
0.61
0.57
0.73
0.03
0.01
0.02
0.02
0.01
AD-53097.1
0.37
0.97
0.67
0.82
0.86
0.01
0.01
0.01
0.02
0.01
AD-53098.1
0.06
0.65
0.22
0.30
0.43
0.00
0.03
0.03
0.00
0.01
AD-53099.1
0.34
0.99
0.61
0.81
0.91
0.00
0.00
0.04
0.02
0.06
AD-53100.1
0.31
1.04
0.95
1.03
1.00
0.02
0.01
0.06
0.02
0.17
AD-53101.1
0.46
0.93
0.63
0.69
0.78
0.00
0.01
0.04
0.03
0.04
AD-53102.1
0.23
0.80
0.60
0.55
0.66
0.00
0.03
0.01
0.02
0.03
AD-53103.1
0.05
0.61
0.27
0.32
0.50
0.01
0.02
0.00
0.01
0.00
AD-53104.1
0.13
0.80
0.64
0.68
0.77
0.00
0.02
0.03
0.01
0.05
AD-53105.1
0.15
0.77
0.43
0.65
0.77
0.01
0.03
0.02
0.02
0.05
AD-53106.1
0.16
0.87
0.72
0.70
0.83
0.01
0.02
0.00
0.00
0.04
AD-53107.1
0.19
0.95
0.62
0.65
0.90
0.00
0.02
0.01
0.03
0.04
AD-53108.1
0.22
0.94
0.60
0.68
0.81
0.00
0.01
0.00
0.03
0.04
AD-53109.1
0.16
1.01
0.82
0.78
0.96
0.01
0.08
0.04
0.01
0.07
AD-53110.1
0.10
0.86
0.79
0.77
0.94
0.00
0.05
0.03
0.01
0.05
AD-53111.1
0.22
0.78
0.94
0.85
1.04
0.01
0.01
0.01
0.01
0.07
AD-53112.1
0.09
0.96
0.64
0.65
0.86
0.01
0.02
0.07
0.07
0.00
AD-53113.1
0.10
0.97
0.71
0.77
0.88
0.01
0.05
0.01
0.02
0.01
AD-53114.1
0.19
0.83
0.48
0.52
0.66
0.01
0.01
0.02
0.01
0.00
AD-53115.1
0.10
0.59
0.42
0.44
0.66
0.01
0.03
0.04
0.00
0.02
AD-53116.1
0.11
0.87
0.82
0.85
0.95
0.00
0.05
0.05
0.05
0.05
AD-53117.1
0.52
0.64
1.21
1.00
1.08
0.01
0.03
0.09
0.04
0.07
AD-53118.1
0.19
1.04
0.60
0.72
0.94
0.00
0.07
0.02
0.05
0.06
AD-53119.1
0.06
0.77
0.44
0.47
0.64
0.01
0.03
0.00
0.01
0.01
AD-53120.1
0.10
0.97
0.78
0.89
1.01
0.01
0.04
0.05
0.01
0.04
AD-53121.1
0.23
0.80
0.58
0.69
0.90
0.01
0.02
0.04
0.02
0.06
AD-53122.1
0.09
0.80
0.90
0.94
1.09
0.01
0.07
0.02
0.04
0.10
AD-53123.1
0.27
0.74
0.95
0.93
0.97
0.00
0.01
0.03
0.01
0.08
AD-53124.1
0.08
0.81
0.33
0.34
0.61
0.01
0.02
0.00
0.01
0.01
AD-53125.1
0.08
0.82
0.34
0.38
0.58
0.00
0.02
0.00
0.01
0.07
AD-53126.1
0.15
0.95
0.70
0.86
1.06
0.01
0.04
0.05
0.02
0.00
AD-53127.1
0.21
0.81
0.62
0.75
0.91
0.02
0.04
0.01
0.03
0.00
AD-53128.1
0.08
0.79
0.80
1.14
1.09
0.00
0.06
0.04
0.01
0.03
AD-53129.1
0.48
0.78
1.05
1.00
1.10
0.00
0.01
0.06
0.01
0.03
AD-53130.1
0.25
1.08
0.63
0.72
0.88
0.01
0.02
0.00
0.01
0.00
AD-53131.1
0.14
0.96
0.54
0.57
0.81
0.02
0.02
0.05
0.01
0.04
AD-53132.1
0.03
0.54
0.24
0.27
0.49
0.00
0.02
0.02
0.00
0.01
AD-53133.1
0.12
0.76
0.50
0.67
0.93
0.00
0.03
0.01
0.01
0.06
AD-53134.1
0.28
0.86
1.14
0.81
0.97
0.01
0.04
0.05
0.02
0.04
AD-53135.1
0.47
0.74
1.03
0.94
1.09
0.01
0.03
0.04
0.07
0.04
AD-53136.1
0.09
0.99
0.64
0.69
0.94
0.01
0.05
0.01
0.05
0.02
AD-53137.1
0.08
0.75
0.39
0.39
0.59
0.01
0.03
0.00
0.00
0.00
AD-53138.1
0.04
0.71
0.33
0.34
0.60
0.00
0.02
0.00
0.03
0.00
AD-53139.1
0.11
0.76
0.55
0.66
0.84
0.01
0.01
0.06
0.01
0.02
AD-53140.1
0.09
0.71
0.64
0.71
0.86
0.00
0.04
0.01
0.02
0.02
AD-53141.1
0.24
1.09
0.77
0.91
0.93
0.00
0.01
0.00
0.06
0.00
AD-53142.1
0.13
0.95
0.55
0.70
0.82
0.01
0.03
0.03
0.04
0.02
AD-53143.1
0.13
0.91
0.67
0.83
0.94
0.01
0.00
0.03
0.03
0.07
AD-53144.1
0.10
0.72
0.54
0.69
0.84
0.01
0.03
0.01
0.03
0.00
AD-53145.1
0.08
0.72
0.70
0.78
0.88
0.01
0.03
0.01
0.08
0.02
AD-53146.1
0.83
1.07
0.85
0.96
0.98
0.01
0.06
0.00
0.05
0.00
AD-53147.1
0.08
0.56
0.27
0.34
0.47
0.00
0.01
0.01
0.01
0.01
AD-53148.1
0.06
0.81
0.61
0.68
0.74
0.01
0.00
0.03
0.06
0.05
AD-53149.1
0.23
0.86
0.71
0.83
0.92
0.01
0.02
0.06
0.02
0.03
AD-53150.1
0.41
0.70
1.03
1.09
1.03
0.03
0.06
0.03
0.04
0.01
TABLE 12
Dose response screen results for ANGPTL3
GalNac-conjugated dsRNA sequences
A subset of active siRNAs from the single dose screen (refer to
data in Table 11) was tested in a dose response experiment by
free uptake in PCH cells. A subset of these active siRNAs was
also tested in dose response in Hep3B cells by transfection.
IC50 (nM)
Free uptake
Transfection (RNAiMax)
AD-53063.1
1.60
0.03
AD-53001.1
2.27
0.01
AD-53015.1
2.90
0.02
AD-52953.1
2.94
0.03
AD-52986.1
3.30
0.03
AD-53024.1
3.42
0.02
AD-53033.1
3.42
0.02
AD-53027.1
3.84
0.01
AD-53030.1
3.90
0.03
AD-53080.1
4.08
0.04
AD-53073.1
4.20
0.05
AD-52965.1
4.63
ND
AD-53092.1
5.37
ND
AD-53132.1
5.54
ND
AD-52983.1
5.55
ND
AD-52954.1
5.67
ND
AD-52961.1
6.37
ND
AD-52994.1
6.43
ND
AD-53098.1
6.58
ND
AD-52970.1
6.71
ND
AD-53075.1
6.74
ND
AD-53086.1
7.08
ND
AD-52971.1
7.50
ND
AD-53064.1
8.33
ND
AD-53147.1
8.34
ND
AD-52969.1
8.86
ND
AD-53077.1
8.98
ND
AD-52981.1
9.44
ND
AD-52977.1
10.45
ND
AD-53071.1
11.19
ND
AD-52960.1
13.03
ND
AD-53095.1
21.31
ND
AD-53103.1
21.92
ND
TABLE 13
Results of single dose screen using sequences listed in Table 10.
10
0.1
0.025
STDEV
STDEV
STDEV
Duplex
nM
nM
nM
10 nM
0.1 nM
0.025 nM
AD-52719.1
0.01
0.60
0.35
0.000
0.093
0.002
AD-52717.1
0.02
0.31
0.32
0.001
0.014
0.008
AD-52713.1
0.02
0.37
0.36
0.001
0.011
0.007
AD-52711.1
0.03
0.22
0.23
0.005
0.011
0.009
AD-52718.1
0.03
0.31
0.39
0.000
0.025
0.023
AD-52687.1
0.03
0.37
0.38
0.005
0.020
0.002
AD-52699.1
0.03
0.25
0.21
0.002
0.011
0.002
AD-52679.1
0.03
0.51
0.24
0.345
0.008
AD-52689.1
0.03
0.44
0.42
0.000
0.039
0.002
AD-52700.1
0.03
0.56
0.57
0.005
0.044
0.020
AD-52637.1
0.04
0.27
0.23
0.001
0.003
0.005
AD-52730.1
0.04
0.61
0.59
0.005
0.053
0.014
AD-52725.1
0.04
0.62
0.61
0.002
0.027
0.012
AD-52688.1
0.04
0.23
0.20
0.006
0.012
0.011
AD-52661.1
0.04
0.61
0.25
0.001
0.449
0.009
AD-52667.1
0.04
0.28
0.22
0.004
0.018
0.013
AD-52665.1
0.04
0.43
0.48
0.007
0.019
0.009
AD-52638.1
0.04
0.28
0.25
0.000
0.016
0.027
AD-52724.1
0.05
0.86
0.76
0.001
0.055
0.011
AD-52705.1
0.05
0.74
0.65
0.004
0.022
0.016
AD-52708.1
0.05
0.53
0.52
0.001
0.034
0.013
AD-52659.1
0.05
0.56
0.48
0.000
0.000
0.033
AD-52678.1
0.05
0.53
0.53
0.002
0.034
0.000
AD-52670.1
0.05
0.35
0.33
0.002
0.009
0.003
AD-52695.1
0.05
0.63
0.67
0.001
0.012
0.013
AD-52704.1
0.05
0.55
0.53
0.002
0.005
0.034
AD-52683.1
0.05
0.36
0.28
0.002
0.021
0.011
AD-52673.1
0.05
0.22
0.19
0.023
0.010
0.002
AD-52721.1
0.05
0.60
0.53
0.003
0.006
0.029
AD-52710.1
0.05
0.56
0.40
0.007
0.073
0.000
AD-52714.1
0.05
0.40
0.51
0.000
0.016
0.003
AD-52686.1
0.05
0.57
0.60
0.003
0.014
0.000
AD-52645.1
0.05
0.62
0.59
0.004
0.030
0.003
AD-52662.1
0.05
0.55
0.52
0.002
0.030
0.008
AD-52720.1
0.05
0.50
0.46
0.003
0.007
0.011
AD-52654.1
0.05
0.29
0.36
0.008
0.037
0.014
AD-52680.1
0.06
0.48
0.41
0.001
0.019
0.026
AD-52723.1
0.06
0.84
0.76
0.001
0.041
0.004
AD-52726.1
0.06
0.72
0.66
0.003
0.028
0.016
AD-52701.1
0.06
0.67
0.39
0.001
0.003
0.002
AD-52694.1
0.06
0.68
0.59
0.004
0.040
0.012
AD-52685.1
0.06
0.30
0.25
0.002
0.013
0.016
AD-52728.1
0.06
0.80
0.79
0.005
0.043
0.015
AD-52676.1
0.06
0.68
0.67
0.002
0.023
0.029
AD-52639.1
0.06
0.47
0.45
0.000
0.005
0.007
AD-52722.1
0.06
0.81
0.93
0.005
0.004
0.027
AD-52682.1
0.06
0.87
0.73
0.009
0.038
0.014
AD-52660.1
0.07
0.69
0.68
0.002
0.014
0.017
AD-52709.1
0.07
0.89
0.82
0.001
0.013
0.020
AD-52643.1
0.07
0.27
0.24
0.006
0.016
0.012
AD-52696.1
0.07
0.53
0.46
0.003
0.026
0.007
AD-52657.1
0.08
0.60
0.58
0.008
0.030
0.006
AD-52706.1
0.08
0.84
0.78
0.001
0.021
0.019
AD-52653.1
0.08
0.41
0.45
0.057
0.004
0.029
AD-52656.1
0.08
0.65
0.50
0.004
0.022
0.012
AD-52693.1
0.09
0.61
0.62
0.007
0.021
0.018
AD-52692.1
0.09
0.54
0.52
0.023
0.018
0.033
AD-52674.1
0.10
0.79
0.64
0.001
0.008
0.028
AD-52648.1
0.10
0.67
0.53
0.002
0.013
0.028
AD-52651.1
0.10
0.84
0.73
0.000
0.000
0.007
AD-52641.1
0.10
0.62
0.50
0.004
0.172
0.002
AD-52707.1
0.10
0.92
0.81
0.001
0.018
0.032
AD-52671.1
0.11
0.87
0.84
0.005
0.034
0.025
AD-52650.1
0.12
0.88
0.94
0.007
0.013
0.041
AD-52642.1
0.12
0.90
0.76
0.015
0.022
0.004
AD-52675.1
0.13
0.94
0.89
0.001
0.018
0.044
AD-52647.1
0.13
0.80
0.79
0.031
0.008
0.023
AD-52716.1
0.14
0.61
0.69
0.010
0.060
0.013
AD-52649.1
0.14
0.31
0.29
0.136
0.020
0.006
AD-52677.1
0.16
1.01
0.72
0.059
0.040
0.007
AD-52697.1
0.16
0.86
0.77
0.012
0.021
0.015
AD-52715.1
0.17
0.90
0.89
0.005
0.009
0.022
AD-52691.1
0.18
0.93
0.88
0.004
0.036
0.017
AD-52698.1
0.20
0.97
0.87
0.010
0.028
0.000
AD-52672.1
0.20
0.70
0.66
0.170
0.014
0.019
AD-52712.1
0.29
0.92
0.90
0.007
0.036
0.004
AD-52690.1
0.30
0.95
0.85
0.115
0.032
0.004
AD-52640.1
0.30
1.04
0.91
0.018
0.046
0.013
AD-52684.1
0.31
0.90
0.94
0.014
0.018
0.014
AD-52666.1
0.32
1.04
0.91
0.013
0.005
0.004
AD-52703.1
0.32
1.02
0.96
0.016
0.015
0.005
AD-52729.1
0.33
1.02
0.87
0.032
0.020
0.008
AD-52668.1
0.35
0.94
0.90
0.029
0.046
0.026
AD-52681.1
0.57
1.00
0.99
0.003
0.034
0.039
AD-52702.1
0.72
1.02
0.92
0.658
0.060
0.014
AD-52727.1
0.73
1.03
0.91
0.004
0.065
0.027
AD-52663.1
0.78
1.05
0.96
0.027
0.010
0.005
AD-52669.1
0.91
0.91
0.94
0.004
0.049
0.032
AD-1955
0.95
0.84
0.95
0.005
0.021
0.019
AD-1955
0.97
1.07
1.03
0.000
0.021
0.015
AD-1955
1.01
1.08
1.01
0.035
0.011
0.005
mock
1.02
0.96
0.97
0.030
0.037
0.005
AD-1955
1.08
1.03
1.02
0.032
0.051
0.005
AD-52652.1
1.13
1.11
1.02
0.028
0.043
0.020
AD-52658.1
1.33
1.10
0.93
0.091
0.043
0.018
AD-52664.1
1.49
0.95
0.88
0.438
0.019
0.009
AD-52752.1
0.03
0.43
0.69
0.002
0.015
0.017
AD-52741.1
0.03
0.56
0.86
0.001
0.044
0.021
AD-52804.1
0.03
0.49
0.89
0.001
0.002
0.017
AD-52764.1
0.03
0.54
0.79
0.005
0.016
0.078
AD-52770.1
0.03
0.58
0.78
0.000
0.006
0.027
AD-52735.1
0.03
0.31
0.46
0.003
0.031
0.009
AD-52810.1
0.03
0.67
0.86
0.001
0.013
0.025
AD-52759.1
0.03
0.54
0.79
0.000
0.018
0.023
AD-52736.1
0.03
0.51
0.60
0.004
0.012
0.023
AD-52775.1
0.03
0.54
0.73
0.005
0.024
0.022
AD-52758.1
0.03
0.57
0.78
0.001
0.014
0.050
AD-52743.1
0.03
0.45
0.67
0.002
0.018
0.033
AD-52747.1
0.04
0.57
0.84
0.002
0.061
0.058
AD-52819.1
0.04
0.26
0.45
0.005
0.001
0.022
AD-52765.1
0.04
0.68
0.83
0.000
0.013
0.053
AD-52754.1
0.04
0.76
1.00
0.000
0.007
0.015
AD-52787.1
0.05
0.55
0.68
0.001
0.043
0.060
AD-52791.1
0.05
0.70
0.91
0.001
0.014
0.084
AD-52811.1
0.05
0.73
0.84
0.002
0.014
0.058
AD-52817.1
0.05
0.77
0.92
0.003
0.011
0.031
AD-52745.1
0.06
0.62
0.77
0.007
0.021
0.000
AD-52749.1
0.06
0.63
0.88
0.005
0.037
0.043
AD-52740.1
0.06
0.83
0.94
0.007
0.012
0.051
AD-52796.1
0.06
0.72
0.92
0.003
0.021
0.054
AD-52820.1
0.06
0.90
0.87
0.001
0.026
0.064
AD-52809.1
0.06
0.76
0.90
0.001
0.037
0.027
AD-52760.1
0.06
0.81
0.97
0.001
0.056
0.047
AD-52767.1
0.07
0.55
0.55
0.001
0.016
0.013
AD-52734.1
0.07
0.61
0.64
0.004
0.003
0.003
AD-52794.1
0.07
0.94
0.87
0.007
0.014
0.051
AD-52797.1
0.07
0.69
0.87
0.004
0.000
0.038
AD-52737.1
0.08
0.70
0.84
0.004
0.031
0.012
AD-52812.1
0.08
0.75
0.88
0.004
0.000
0.056
AD-52748.1
0.08
0.70
0.89
0.001
0.010
0.009
AD-52782.1
0.08
0.68
0.78
0.004
0.023
0.011
AD-52816.1
0.08
0.71
0.88
0.003
0.042
0.060
AD-52763.1
0.08
0.68
0.77
0.002
0.013
0.026
AD-52788.1
0.08
0.89
1.00
0.004
0.017
0.034
AD-52762.1
0.08
0.78
0.91
0.007
0.046
0.009
AD-52785.1
0.08
0.88
0.95
0.002
0.004
0.019
AD-52800.1
0.09
0.82
0.94
0.001
0.040
0.005
AD-52792.1
0.09
0.93
0.94
0.002
0.018
0.037
AD-52784.1
0.10
0.84
0.92
0.000
0.066
0.032
AD-52746.1
0.10
0.82
0.93
0.002
0.060
0.059
AD-52814.1
0.10
0.85
0.88
0.002
0.042
0.013
AD-52751.1
0.10
0.88
0.98
0.005
0.030
0.067
AD-52786.1
0.10
0.81
0.81
0.006
0.028
0.048
AD-52755.1
0.10
0.93
0.99
0.003
0.032
0.048
AD-52808.1
0.11
0.98
0.92
0.000
0.038
0.032
AD-52815.1
0.11
0.96
0.96
0.002
0.009
0.000
AD-52805.1
0.11
0.79
0.86
0.003
0.050
0.008
AD-52777.1
0.11
0.88
0.94
0.001
0.065
0.000
AD-52756.1
0.11
0.92
0.91
0.003
0.032
0.004
AD-52733.1
0.12
0.66
0.65
0.005
0.071
0.022
AD-52739.1
0.13
0.83
0.95
0.002
0.008
0.061
AD-52780.1
0.13
0.70
0.67
0.012
0.021
0.059
AD-52798.1
0.13
0.64
0.97
0.001
0.006
0.038
AD-52776.1
0.14
0.97
0.94
0.011
0.029
0.023
AD-52753.1
0.15
0.88
1.09
0.001
0.048
0.005
AD-52778.1
0.16
0.76
0.69
0.003
0.067
0.003
AD-52744.1
0.16
0.90
0.91
0.002
0.000
0.049
AD-52750.1
0.16
0.87
1.01
0.000
0.060
0.055
AD-52774.1
0.17
0.71
0.89
0.002
0.010
0.017
AD-52803.1
0.18
0.87
0.92
0.015
0.026
0.040
AD-52821.1
0.18
0.86
0.87
0.005
0.046
0.055
AD-52781.1
0.18
0.78
0.66
0.008
0.000
0.023
AD-52779.1
0.20
0.83
0.66
0.002
0.024
0.016
AD-52793.1
0.20
0.74
0.88
0.010
0.025
0.069
AD-52799.1
0.20
0.75
1.01
0.005
0.018
0.010
AD-52761.1
0.22
0.83
0.92
0.000
0.024
0.023
AD-52768.1
0.22
0.96
0.97
0.001
ND
0.028
AD-52757.1
0.23
1.02
0.95
0.018
0.040
0.042
AD-52806.1
0.24
0.96
0.87
0.011
0.084
0.055
AD-52771.1
0.25
0.92
0.98
0.010
0.018
0.048
AD-52802.1
0.30
0.95
1.00
0.010
0.019
0.005
AD-52731.1
0.30
0.85
0.75
0.001
0.067
0.022
AD-52813.1
0.30
1.07
0.98
0.001
0.109
0.014
AD-52742.1
0.31
0.95
1.03
0.005
0.028
0.056
AD-52766.1
0.35
0.97
1.00
0.010
0.024
0.044
AD-52732.1
0.41
0.79
0.73
0.004
0.016
0.039
AD-52773.1
0.43
0.99
0.92
0.004
0.029
0.022
AD-52772.1
0.43
1.00
1.02
0.006
0.000
0.065
AD-52822.1
0.44
0.68
0.81
0.004
0.010
0.016
AD-52783.1
0.45
0.66
0.76
0.009
0.036
0.019
AD-52789.1
0.50
0.68
0.78
0.010
0.053
0.004
AD-52795.1
0.50
0.82
0.69
0.000
0.080
0.054
AD-52801.1
0.54
0.70
0.79
0.018
0.038
0.035
AD-52807.1
0.57
0.76
0.93
0.006
0.011
0.032
AD-52769.1
0.76
0.97
0.92
0.015
0.085
0.045
AD-1955
0.90
0.96
1.04
0.018
0.165
0.010
AD-52818.1
0.92
1.03
0.92
0.009
0.010
0.063
AD-1955
1.01
0.90
0.96
0.005
0.031
0.019
AD-1955
1.05
1.09
1.00
0.046
0.085
0.005
AD-1955
1.05
1.07
1.00
0.010
0.031
0.039
mock
1.20
0.98
0.92
0.000
0.014
0.005
mock
1.25
0.99
1.00
0.006
0.005
0.034
TABLE 14
Results of a dose response screen using
a subset of sequences from Table 13.
A subset of active ANGPTL3 siRNAs from Table 10 were
tested by transfection in Hep3B cells in dose response screens.
Duplex
IC50 (nM)
AD-52819.1
0.0036
AD-52667.1
0.0037
AD-52638.1
0.0048
AD-52673.1
0.0049
AD-52711.1
0.0050
AD-52661.1
0.0054
AD-52654.1
0.0058
AD-52637.1
0.0058
AD-52643.1
0.0060
AD-52685.1
0.0062
AD-52670.1
0.0064
AD-52679.1
0.0064
AD-52649.1
0.0066
AD-52683.1
0.0069
AD-52688.1
0.0071
AD-52717.1
0.0072
AD-52699.1
0.0073
AD-52714.1
0.0086
AD-52718.1
0.0088
AD-52735.1
0.0093
AD-52653.1
0.0102
AD-52687.1
0.0109
AD-52680.1
0.0120
AD-52713.1
0.0133
AD-52720.1
0.0143
AD-52639.1
0.0161
AD-52696.1
0.0163
AD-52662.1
0.0179
AD-52659.1
0.0180
AD-52710.1
0.0195
AD-52689.1
0.0216
AD-52787.1
0.0242
AD-52765.1
0.0318
TABLE 15
IDs of duplex pairs for which both an unconjuaged and a
GalNac-conjugated version were synthesized and tested
These duplexes have the same sequence and modification pattern.
Unconjugated duplex ID
GalNac conjugated duplex ID
AD-52637.1
AD-52953.1
AD-52638.1
AD-52954.1
AD-52639.1
AD-52955.1
AD-52640.1
AD-52956.1
AD-52641.1
AD-52957.1
AD-52642.1
AD-52958.1
AD-52643.1
None
None
AD-52960.1
None
AD-52961.1
AD-52645.1
AD-52962.1
AD-52647.1
AD-52963.1
AD-52648.1
AD-52964.1
AD-52649.1
AD-52965.1
AD-52650.1
AD-52966.1
AD-52651.1
AD-52967.1
AD-52652.1
AD-52968.1
AD-52653.1
AD-52969.1
AD-52654.1
AD-52970.1
None
AD-52971.1
AD-52656.1
AD-52972.1
AD-52657.1
AD-52973.1
AD-52658.1
AD-52974.1
AD-52659.1
AD-52975.1
AD-52660.1
AD-52976.1
AD-52661.1
AD-52977.1
AD-52662.1
AD-52978.1
AD-52663.1
AD-52979.1
AD-52664.1
AD-52980.1
AD-52665.1
AD-52981.1
AD-52666.1
AD-52982.1
AD-52667.1
AD-52983.1
AD-52668.1
AD-52984.1
AD-52669.1
AD-52985.1
AD-52670.1
AD-52986.1
AD-52671.1
AD-52987.1
AD-52672.1
AD-52988.1
AD-52673.1
AD-52989.1
AD-52674.1
AD-52990.1
AD-52675.1
AD-52991.1
AD-52676.1
AD-52992.1
AD-52677.1
AD-52993.1
AD-52678.1
AD-52994.1
AD-52679.1
AD-52995.1
AD-52680.1
AD-52996.1
AD-52681.1
AD-52997.1
AD-52682.1
AD-52998.1
AD-52683.1
AD-52999.1
AD-52684.1
AD-53000.1
AD-52685.1
AD-53001.1
AD-52686.1
AD-53002.1
AD-52687.1
AD-53003.1
AD-52688.1
AD-53004.1
AD-52689.1
AD-53005.1
AD-52690.1
AD-53006.1
AD-52691.1
AD-53007.1
AD-52692.1
AD-53008.1
AD-52693.1
AD-53009.1
AD-52694.1
AD-53010.1
AD-52695.1
AD-53011.1
AD-52696.1
AD-53012.1
AD-52697.1
AD-53013.1
AD-52698.1
AD-53014.1
AD-52699.1
AD-53015.1
AD-52700.1
AD-53016.1
AD-52701.1
AD-53017.1
AD-52702.1
AD-53018.1
AD-52703.1
AD-53019.1
AD-52704.1
AD-53020.1
AD-52705.1
AD-53021.1
AD-52706.1
AD-53022.1
AD-52707.1
AD-53023.1
AD-52708.1
AD-53024.1
AD-52709.1
AD-53025.1
AD-52710.1
AD-53026.1
AD-52711.1
AD-53027.1
AD-52712.1
AD-53028.1
AD-52713.1
AD-53029.1
AD-52714.1
AD-53030.1
AD-52715.1
AD-53031.1
AD-52716.1
AD-53032.1
AD-52717.1
AD-53033.1
AD-52718.1
AD-53034.1
AD-52719.1
AD-53035.1
AD-52720.1
AD-53036.1
AD-52721.1
AD-53037.1
AD-52722.1
AD-53038.1
AD-52723.1
AD-53039.1
AD-52724.1
AD-53040.1
AD-52725.1
AD-53041.1
AD-52726.1
AD-53042.1
AD-52727.1
AD-53043.1
AD-52728.1
AD-53044.1
AD-52729.1
AD-53045.1
AD-52730.1
AD-53046.1
AD-52731.1
AD-53059.1
AD-52732.1
AD-53060.1
AD-52733.1
AD-53061.1
AD-52734.1
AD-53062.1
AD-52735.1
AD-53063.1
AD-52736.1
AD-53064.1
AD-52737.1
AD-53065.1
None
AD-53066.1
AD-52739.1
AD-53067.1
AD-52740.1
AD-53068.1
AD-52741.1
AD-53069.1
AD-52742.1
AD-53070.1
AD-52743.1
AD-53071.1
AD-52744.1
AD-53072.1
AD-52745.1
AD-53073.1
AD-52746.1
AD-53074.1
AD-52747.1
AD-53075.1
AD-52748.1
AD-53076.1
AD-52749.1
AD-53077.1
AD-52750.1
AD-53078.1
AD-52751.1
AD-53079.1
AD-52752.1
AD-53080.1
AD-52753.1
AD-53081.1
AD-52754.1
AD-53082.1
AD-52755.1
AD-53083.1
AD-52756.1
AD-53084.1
AD-52757.1
AD-53085.1
AD-52758.1
AD-53086.1
AD-52759.1
AD-53087.1
AD-52760.1
AD-53088.1
AD-52761.1
AD-53089.1
AD-52762.1
AD-53090.1
AD-52763.1
AD-53091.1
AD-52764.1
AD-53092.1
AD-52765.1
AD-53093.1
AD-52766.1
AD-53094.1
AD-52767.1
AD-53095.1
AD-52768.1
AD-53096.1
AD-52769.1
AD-53097.1
AD-52770.1
AD-53098.1
AD-52771.1
AD-53099.1
AD-52772.1
AD-53100.1
AD-52773.1
AD-53101.1
AD-52774.1
AD-53102.1
AD-52775.1
AD-53103.1
AD-52776.1
AD-53104.1
AD-52777.1
AD-53105.1
AD-52778.1
AD-53106.1
AD-52779.1
AD-53107.1
AD-52780.1
AD-53108.1
AD-52781.1
AD-53109.1
AD-52782.1
AD-53110.1
AD-52783.1
AD-53111.1
AD-52784.1
AD-53112.1
AD-52785.1
AD-53113.1
AD-52786.1
AD-53114.1
AD-52787.1
AD-53115.1
AD-52788.1
AD-53116.1
AD-52789.1
AD-53117.1
None
AD-53118.1
AD-52791.1
AD-53119.1
AD-52792.1
AD-53120.1
AD-52793.1
AD-53121.1
AD-52794.1
AD-53122.1
AD-52795.1
AD-53123.1
AD-52796.1
AD-53124.1
AD-52797.1
AD-53125.1
AD-52798.1
AD-53126.1
AD-52799.1
AD-53127.1
AD-52800.1
AD-53128.1
AD-52801.1
AD-53129.1
AD-52802.1
AD-53130.1
AD-52803.1
AD-53131.1
AD-52804.1
AD-53132.1
AD-52805.1
AD-53133.1
AD-52806.1
AD-53134.1
AD-52807.1
AD-53135.1
AD-52808.1
AD-53136.1
AD-52809.1
AD-53137.1
AD-52810.1
AD-53138.1
AD-52811.1
AD-53139.1
AD-52812.1
AD-53140.1
AD-52813.1
AD-53141.1
AD-52814.1
AD-53142.1
AD-52815.1
AD-53143.1
AD-52816.1
AD-53144.1
AD-52817.1
AD-53145.1
AD-52818.1
AD-53146.1
AD-52819.1
AD-53147.1
AD-52820.1
AD-53148.1
AD-52821.1
AD-53149.1
AD-52822.1
AD-53150.1
In Vivo Tests
Example 3
Test Articles
In vivo experiments were conducted using dsRNA sequences of the invention. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). The dsRNA sequence used as a negative control was luciferase-conjugated AD-48399B1 (“Luc”, sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). Also used as a negative control was GalNal-conjugated AD-1955 containing alternating 2′-methyl and 2′ fluoro modifications.
Experimental Procedure
The dsRNA sequences were tested in C57BL/6 (WT) and ob/ob mice. WT mice received five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5 or 20 mg/kg; and ob/ob mice received five daily doses of NPLs formulated with Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles were administered by subcutaneous injection according to the procedure shown in FIG. 1. Specifically, five daily doses of the test articles were administered on five consecutive days (day 0, 1, 2, 3 and 4), and blood samples were collected 5, 3 or 1 day prior to administration, as well as on days 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45 and 50 post-administration. The collected blood samples were used to measure the expression of ANGPTL3 protein using an ELISA assay. Levels of serum triglycerides (TGs), low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc) and total cholesterol (TC) were also measured using an Olympus Analyzer.
Results
Shown in FIG. 2, Panel A, are levels of murine ANGPTL3 (mANGPTL3, protein measured in WT mice after administration of control or ANG at 5 or 20 mg/kg. Also shown in FIG. 2, Panel B are levels of mANGPTL3 protein measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that, for both WT and ob/ob mice, administration of ANG results in decreased levels of mANGPTL3 protein, as compared to controls.
Shown in FIG. 3, Panel A, are levels of LDL-c measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 3, Panel B are levels of LDL-c measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of LDL-c, particularly in ob/ob mice, as compared to controls.
Shown in FIG. 4, Panel A, are levels of triglycerides measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 4, Panel B are levels of triglycerides measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of tryglycerides, particularly, in ob/ob mice, as compared to controls.
Shown in FIG. 5, Panel A and B are levels of total cholesterol (TC) measured in WT and ob/ob mice, respectively, after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes a moderate decrease in TC levels in ob/ob mice, but not in WT mice. Similarly, administration of ANG causes a moderate decrease in HDL-c levels in ob/ob mice, but not in WT mice, as is shown in the graphs in FIG. 6.
Example 4
Test Article
The effect of a single injection of dsRNA sequence of the invention on the level of ANGPTL3 protein was tested. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). PBS was used as a negative control.
Experimental Procedure
The dsRNA sequences were tested in Human PCS Transgenic mouse characterized by liver-specific expression of full-length human PCSK9 gene. Human PCS transgenic mice were dosed with the AD-52981 or PBS using a single subcutaneous injection. The mice were divided into four groups, each group consisting of two males and two females. Each group received an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose of AD-52981. Blood samples were collected at day 1 and day 0 prior to dosing, and at 72 hours post dosing. ANGPTL3 protein levels were measured by ELISA and compared to levels at day 1 and day 0 prior to dosing.
Results
Shown in FIG. 7, are levels of murine ANGPTL3 protein (mANGPTL3) measured in Human PCS transgenic mice. The data shown is expressed relative to PBS control and represents an average for 2 males and 2 females in each group. Error bars represent standard deviation. The data indicates that administration of a single injection of AD-52981 reduces the levels of ANGPTL3 protein in the mice in a dose-dependent manner, with the dose of 60 mg/kg decreasing the levels of ANGPTL3 protein more than five-fold (see FIG. 7).
SEQUENCES
>gi|41327750|ref|NM_014495.21 Homo sapiens
angiopoietin-like 3 (ANGPTL3), mRNA
SEQ ID NO: 1
TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATT
GAAAATCAAGATAAAAATGTTCACAATTAAGCTCC
TTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGA
ATTGATCAAGACAATTCATCATTTGATTCTCTATC
TCCAGAGCCAAAATCAAGATTTGCTATGTTAGACG
ATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTG
GGACATGGTCTTAAAGACTTTGTCCATAAGACGAA
GGGCCAAATTAATGACATATTTCAAAAACTCAACA
TATTTGATCAGTCTTTTTATGATCTATCGCTGCAA
ACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAG
AAGAACTACATATAAACTACAAGTCAAAAATGAAG
AGGTAAAGAATATGTCACTTGAACTCAACTCAAAA
CTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCA
ACAAAAAGTGAAATATTTAGAAGAGCAACTAACTA
ACTTAATTCAAAATCAACCTGAAACTCCAGAACAC
CCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAA
ACAAGATAATAGCATCAAAGACCTTCTCCAGACCG
TGGAAGACCAATATAAACAATTAAACCAACAGCAT
AGTCAAATAAAAGAAATAGAAAATCAGCTCAGAAG
GACTAGTATTCAAGAACCCACAGAAATTTCTCTAT
CTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTT
CTTCAGTTGAATGAAATAAGAAATGTAAAACATGA
TGGCATTCCTGCTGAATGTACCACCATTTATAACA
GAGGTGAACATACAAGTGGCATGTATGCCATCAGA
CCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGA
TGTTATATCAGGTAGTCCATGGACATTAATTCAAC
ATCGAATAGATGGATCACAAAACTTCAATGAAACG
TGGGAGAACTACAAATATGGTTTTGGGAGGCTTGA
TGGAGAATTTTGGTTGGGCCTAGAGAAGATATACT
CCATAGTGAAGCAATCTAATTATGTTTTACGAATT
GAGTTGGAAGACTGGAAAGACAACAAACATTATAT
TGAATATTCTTTTTACTTGGGAAATCACGAAACCA
ACTATACGCTACATCTAGTTGCGATTACTGGCAAT
GTCCCCAATGCAATCCCGGAAAACAAAGATTTGGT
GTTTTCTACTTGGGATCACAAAGCAAAAGGACACT
TCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGG
TGGCATGATGAGTGTGGAGAAAACAACCTAAATGG
TAAATATAACAAACCAAGAGCAAAATCTAAGCCAG
AGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAAT
GGAAGGTTATACTCTATAAAATCAACCAAAATGTT
GATCCATCCAACAGATTCAGAAAGCTTTGAATGAA
CTGAGGCAAATTTAAAAGGCAATAATTTAAACATT
AACCTCATTCCAAGTTAATGTGGTCTAATAATCTG
GTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGA
TTTTTTTTATCTTAAAGTCACTGTCTATTTAAGAT
TAAACATACAATCACATAACCTTAAAGAATACCGT
TTACATTTCTCAATCAAAATTCTTATAATACTATT
TGTTTTAAATTTTGTGATGTGGGAATCAATTTTAG
ATGGTCACAATCTAGATTATAATCAATAGGTGAAC
TTATTAAATAACTTTTCTAAATAAAAAATTTAGAG
ACTTTTATTTTAAAAGGCATCATATGAGCTAATAT
CACAACTTTCCCAGTTTAAAAAACTAGTACTCTTG
TTAAAACTCTAAACTTGACTAAATACAGAGGACTG
GTAATTGTACAGTTCTTAAATGTTGTAGTATTAAT
TTCAAAACTAAAAATCGTCAGCACAGAGTATGTGT
AAAAATCTGTAATACAAATTTTTAAACTGATGCTT
CATTTTGCTACAAAATAATTTGGAGTAAATGTTTG
ATATGATTTATTTATGAAACCTAATGAAGCAGAAT
TAAATACTGTATTAAAATAAGTTCGCTGTCTTTAA
ACAAATGGAGATGACTACTAAGTCACATTGACTTT
AACATGAGGTATCACTATACCTTATT
>gi|297278846|ref|XM_001086114.2|
PREDICTED: Macaca mulatta
angiopoietin-like 3 (ANGPTL3), mRNA
SEQ ID NO: 2
ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAG
AAGAACACAGTTCCACGTTGCTTGAAATTGAAAAT
CAGGATAAAAATGTTCACAATTAAGCTCCTTCTTT
TTATTGTTCCTCTAGTTATTTCCTCCAGAATTGAC
CAAGACAATTCATCATTTGATTCTGTATCTCCAGA
GCCAAAATCAAGATTTGCTATGTTAGACGATGTAA
AAATTTTAGCCAATGGCCTCCTTCAGTTGGGACAT
GGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCA
AATTAATGACATATTTCAAAAACTCAACATATTTG
ATCAGTCTTTTTATGATCTATCACTGCAAACCAGT
GAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAAC
TACATATAAACTACAAGTCAAAAATGAAGAGGTAA
AGAATATGTCACTTGAACTCAACTCAAAACTTGAA
AGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAA
AGTGAAATATTTAGAAGAGCAACTAACTAACTTAA
TTCAAAATCAACCTGAAACTCCAGAACATCCAGAA
GTAACTTCACTTAAAAGTTTTGTAGAAAAACAAGA
TAATAGCATCAAAGACCTTCTCCAGACTGTGGAAG
AACAATATAAGCAATTAAACCAACAGCACAGTCAA
ATAAAAGAAATAGAAAATCAGCTCAGAATGACTAA
TATTCAAGAACCCACAGAAATTTCTCTATCTTCCA
AGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAG
CTGAATGAAATAAGAAATGTAAAACATGATGGCAT
TCCTGCTGATTGTACCACCATTTACAATAGAGGTG
AACATATAAGTGGCATGTATGCCATCAGACCCAGC
AACTCTCAAGTTTTTCATGTCTACTGTGATGTTGT
ATCAGGTAAAACCTGTCTAAGGAGAATAGATGGAT
CACAAAACTTCAATGAAACGTGGGAGAACTACAAA
TATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTT
GGGCCTAGAGAAGATATACTCCATAGTGAAGCAAT
CTAATTACGTTTTACGAATTGAGTTGGAAGACTGG
AAAGACAACAAACATTATATTGAATATTCTTTTTA
CTTGGGAAATCACGAAACCAACTATACGCTACATG
TAGTTAAGATTACTGGCAATGTCCCCAATGCAATC
CCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGA
TCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGA
GTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGT
GGAGAAAACAACCTAAATGGTAAATATAACAAACC
AAGAACAAAATCTAAGCCAGAGCGGAGAAGAGGAT
TATCCTGGAAGTCTCAAAATGGAAGGTTATACTCT
ATAAAATCAACCAAAATGTTGATCCATCCAACAGA
TTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAA
AAGGCAATAAATTAAACATTAAACTCATTCCAAGT
TAATGTGGTTTAATAATCTGGTATTAAATCCTTAA
GAGAAGGCTTGAGAAATAGATTTTTTTATCTTAAA
GTCACTGTCAATTTAAGATTAAACATACAATCACA
TAACCTTAAAGAATACCATTTACATTTCTCAATCA
AAATTCCTACAACACTATTTGTTTTATATTTTGTG
ATGTGGGAATCAATTTTAGATGGTCGCAATCTAAA
TTATAATCAACAGGTGAACTTACTAAATAACTTTT
CTAAATAAAAAACTTAGAGACTTTAATTTTAAAAG
TCATCATATGAGCTAATATCACAATTTTCCCAGTT
TAAAAAACTAGTTTTCTTGTTAAAACTCTAAACTT
GACTAAATAAAGAGGACTGATAATTATACAGTTCT
TAAATTTGTTGTAATATTAATTTCAAAACTAAAAA
TTGTCAGCACAGAGTATGTGTAAAAATCTGTAATA
TAAATTTTTAAACTGATGCCTCATTTTGCTACAAA
ATAATCTGGAGTAAATTTTTGATAGGATTTATTTA
TGAAACCTAATGAAGCAGGATTAAATACTGTATTA
AAATAGGTTCGCTGTCTTTTAAACAAATGGAGATG
ATGATTACTAAGTCACATTGACTTTAATATGAGGT
ATCACTATACCTTA
>gi|142388354|ref|NM_013913.3| Mus
musculus angiopoietin-like 3
(Angpt13), mRNA
SEQ ID NO: 3
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAA
TAATTGAGACAAAAAATGCACACAATTAAATTATT
CCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAG
TGGATCCAGACCTTTCATCATTTGATTCTGCACCT
TCAGAGCCAAAATCAAGATTTGCTATGTTGGATGA
TGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGG
GTCATGGACTTAAAGATTTTGTCCATAAGACTAAG
GGACAAATTAACGACATATTTCAGAAGCTCAACAT
ATTTGATCAGTCTTTTTATGACCTATCACTTCGAA
CCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGA
AGAACTACATCTACACTACAAGTTAAAAACGAGGA
GGTGAAGAACATGTCAGTAGAACTGAACTCAAAGC
TTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAA
CACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAA
CTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACC
CAGAAGTAACATCACTCAAAAGTTTTGTAGAACAG
CAAGACAACAGCATAAGAGAACTCCTCCAGAGTGT
GGAAGAACAGTATAAACAATTAAGTCAACAGCACA
TGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAG
ACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTC
TTCTAAATCAAGAGCACCAAGAACTACTCCCCCTC
TTCAACTGAACGAAACAGAAAATACAGAACAAGAT
GACCTTCCTGCCGACTGCTCTGCCGTTTATAACAG
AGGCGAACATACAAGTGGCGTGTACACTATTAAAC
CAAGAAACTCCCAAGGGTTTAATGTCTACTGTGAT
ACCCAATCAGGCAGTCCATGGACATTAATTCAACA
CCGGAAAGATGGCTCACAGGACTTCAACGAAACAT
GGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGAT
GGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGC
TATAGTCCAACAGTCTAACTACATTTTACGACTCG
AGCTACAAGACTGGAAAGACAGCAAGCACTACGTT
GAATACTCCTTTCACCTGGGCAGTCACGAAACCAA
CTACACGCTACATGTGGCTGAGATTGCTGGCAATA
TCCCTGGGGCCCTCCCAGAGCACACAGACCTGATG
TTTTCTACATGGAATCACAGAGCAAAGGGACAGCT
CTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGT
GGAATGACATATGTGGAGAAAACAACCTAAATGGA
AAATACAACAAACCCAGAACCAAATCCAGACCAGA
GAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCA
GAAAGCTCTATGCTATCAAATCATCCAAAATGATG
CTCCAGCCCACCACCTAAGAAGCTTCAACTGAACT
GAGACAAAATAAAAGATCAATAAATTAAATATTAA
AGTCCTCCCGATCACTGTAGTAATCTGGTATTAAA
ATTTTAATGGAAAGCTTGAGAATTGAATTTCAATT
AGGTTTAAACTCATTGTTAAGATCAGATATCACCG
AATCAACGTAAACAAAATTTATC
>gi|68163568|ref|NM_001025065.1| Rattus
norvegicus angiopoietin-like 3
(Angpt13), mRNA
SEQ ID NO: 4
GACGTTCCAAATTGCTTGAAATTGAATAATTGAAA
CAAAAATGCACACAATTAAGCTGCTCCTTTTTGTT
GTTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGA
CCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAA
AATCAAGATTTGCTATGTTGGATGATGTCAAAATT
TTAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCT
TAAAGATTTTGTCCATAAGACAAAGGGACAAATTA
ATGACATATTTCAGAAGCTCAACATATTTGATCAG
TGTTTTTATGACCTATCACTTCAAACCAATGAAAT
CAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACAT
CTAAACTACAAGTTAAAAACGAAGAGGTGAAGAAT
ATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCT
ACTGGAGGAGAAGATGGCGCTCCAACACAGAGTCA
GGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAG
AACCCGCCTGGGGCTCGGGAGCACCCAGAGGTAAC
GTCACTTAAAAGTTTTGTAGAACAGCAAGATAACA
GCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAA
TATAAACAACTAAGTCAACAGCACATTCAGATAAA
AGAAATAGAAAATCAGCTCAGAAAGACTGGCATTC
AAGAACCCACTGAAAATTCTCTTTATTCTAAACCA
AGAGCACCAAGAACTACTCCCCCTCTTCATCTGAA
GGAAGCAAAAAATATAGAACAAGATGATCTGCCTG
CTGACTGCTCTGCCATTTATAACAGAGGTGAACAT
ACAAGTGGCGTGTATACTATTAGACCAAGCAGCTC
TCAAGTGTTTAATGTCTACTGTGACACCCAATCAG
GCACTCCACGGACATTAATTCAACACCGGAAAGAT
GGCTCTCAAAACTTCAACCAAACGTGGGAAAACTA
CGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGA
TTTCCTTGCATCACTCACTTATCTGTTGATTTAAT
AGTATTAGTTGGGTGTGTTGACACAGGCCTGAGAC
CATAGCGCTTTTGGGCAAGGGGGGAGGAGGAGCAG
CAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCA
CACATTGATACTCCTTCTCGACATTAAGAATTATA
AATTAAGCAGCAATTATAAAATGGGCTGTGGAAAT
GTAACAATAAGCAAAAGCAGACCCCAGTCTTCATA
AAACTGATTGGTAAATATTATCCATGATAGCAACT
GCAATGATCTCATTGTACTTATCACTACTGCATGC
CTGCAGTATGCTTGTTGAAACTTAATTCTATAGTT
CATGGTTATCATAAGTCTTATTAAGGAACATAGTA
TACGCCATTGGCTCTAGTGAGGGGCCATGCTACAA
ATGAGCTGCAAAGATAGCAGTATAGAGCTCTTTCA
GTGATATCCTAAGCACAACGTAACACAGGTGAAAT
GGGCTGGAGGCACAGTTGTGGTGGAACACGCGGCC
AGCAGGACACTGGGACTGATCCCCAGCAGCACAAA
GAAAGTGATAGGAACACAGAGCGAGAGTTAGAAGG
GACAGGGTCACCGTCAGAGATACGGTGTCTAACTC
CTGCAACCCTACCTGTAATTATTCCATATTATAAA
CATATACTATATAACTGTGGGTCTCTGCATGTTCT
AGAATATGAATTCTATTTGATTGTTAATCAAAAAA
AAAAAAAAA
Reverse Complement of SEQ ID NO: 1
SEQ ID NO: 5
AATAAGGTATAGTGATACCTCATGTTAAAGTCAAT
GTGACTTAGTAGTCATCTCCATTTGTTTAAAGACA
GCGAACTTATTTTAATACAGTATTTAATTCTGCTT
CATTAGGTTTCATAAATAAATCATATCAAACATTT
ACTCCAAATTATTTTGTAGCAAAATGAAGCATCAG
TTTAAAAATTTGTATTACAGATTTTTACACATACT
CTGTGCTGACGATTTTTAGTTTTGAAATTAATACT
ACAACATTTAAGAACTGTACAATTACCAGTCCTCT
GTATTTAGTCAAGTTTAGAGTTTTAACAAGAGTAC
TAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCT
CATATGATGCCTTTTAAAATAAAAGTCTCTAAATT
TTTTATTTAGAAAAGTTATTTAATAAGTTCACCTA
TTGATTATAATCTAGATTGTGACCATCTAAAATTG
ATTCCCACATCACAAAATTTAAAACAAATAGTATT
ATAAGAATTTTGATTGAGAAATGTAAACGGTATTC
TTTAAGGTTATGTGATTGTATGTTTAATCTTAAAT
AGACAGTGACTTTAAGATAAAAAAAATCTATTTCT
CAAGCTTTCTCTTAAGGATTTAATACCAGATTATT
AGACCACATTAACTTGGAATGAGGTTAATGTTTAA
ATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAA
AGCTTTCTGAATCTGTTGGATGGATCAACATTTTG
GTTGATTTTATAGAGTATAACCTTCCATTTTGAGA
CTTCCAAGATAATCCTCTTCTCCTCTCTGGCTTAG
ATTTTGCTCTTGGTTTGTTATATTTACCATTTAGG
TTGTTTTCTCCACACTCATCATGCCACCACCAGCC
TCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTT
TTGCTTTGTGATCCCAAGTAGAAAACACCAAATCT
TTGTTTTCCGGGATTGCATTGGGGACATTGCCAGT
AATCGCAACTAGATGTAGCGTATAGTTGGTTTCGT
GATTTCCCAAGTAAAAAGAATATTCAATATAATGT
TTGTTGTCTTTCCAGTCTTCCAACTCAATTCGTAA
AACATAATTAGATTGCTTCACTATGGAGTATATCT
TCTCTAGGCCCAACCAAAATTCTCCATCAAGCCTC
CCAAAACCATATTTGTAGTTCTCCCACGTTTCATT
GAAGTTTTGTGATCCATCTATTCGATGTTGAATTA
ATGTCCATGGACTACCTGATATAACATCACAGTAG
ACATGAAAAACTTGAGAGTTGCTGGGTCTGATGGC
ATACATGCCACTTGTATGTTCACCTCTGTTATAAA
TGGTGGTACATTCAGCAGGAATGCCATCATGTTTT
ACATTTCTTATTTCATTCAACTGAAGAAAGGGAGT
AGTTCTTGGTGCTCTTGGCTTGGAAGATAGAGAAA
TTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGC
TGATTTTCTATTTCTTTTATTTGACTATGCTGTTG
GTTTAATTGTTTATATTGGTCTTCCACGGTCTGGA
GAAGGTCTTTGATGCTATTATCTTGTTTTTCTACA
AAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGG
AGTTTCAGGTTGATTTTGAATTAAGTTAGTTAGTT
GCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGA
ATTTTTTCTTCTAGGAGGCTTTCAAGTTTTGAGTT
GAGTTCAAGTGACATATTCTTTACCTCTTCATTTT
TGACTTGTAGTTTATATGTAGTTCTTCTCAGTTCC
TTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGA
TAGATCATAAAAAGACTGATCAAATATGTTGAGTT
TTTGAAATATGTCATTAATTTGGCCCTTCGTCTTA
TGGACAAAGTCTTTAAGACCATGTCCCAACTGAAG
GAGGCCATTGGCTAAAATTTTTACATCGTCTAACA
TAGCAAATCTTGATTTTGGCTCTGGAGATAGAGAA
TCAAATGATGAATTGTCTTGATCAATTCTGGAGGA
AATAACTAGAGGAACAATAAAAAGAAGGAGCTTAA
TTGTGAACATTTTTATCTTGATTTTCAATTTCAAG
CAACGTGGAACTGTTTTCTTCTGGAA
Reverse Complement of SEQ ID NO: 2
SEQ ID NO: 6
TAAGGTATAGTGATACCTCATATTAAAGTCAATGT
GACTTAGTAATCATCATCTCCATTTGTTTAAAAGA
CAGCGAACCTATTTTAATACAGTATTTAATCCTGC
TTCATTAGGTTTCATAAATAAATCCTATCAAAAAT
TTACTCCAGATTATTTTGTAGCAAAATGAGGCATC
AGTTTAAAAATTTATATTACAGATTTTTACACATA
CTCTGTGCTGACAATTTTTAGTTTTGAAATTAATA
TTACAACAAATTTAAGAACTGTATAATTATCAGTC
CTCTTTATTTAGTCAAGTTTAGAGTTTTAACAAGA
AAACTAGTTTTTTAAACTGGGAAAATTGTGATATT
AGCTCATATGATGACTTTTAAAATTAAAGTCTCTA
AGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCA
CCTGTTGATTATAATTTAGATTGCGACCATCTAAA
ATTGATTCCCACATCACAAAATATAAAACAAATAG
TGTTGTAGGAATTTTGATTGAGAAATGTAAATGGT
ATTCTTTAAGGTTATGTGATTGTATGTTTAATCTT
AAATTGACAGTGACTTTAAGATAAAAAAATCTATT
TCTCAAGCCTTCTCTTAAGGATTTAATACCAGATT
ATTAAACCACATTAACTTGGAATGAGTTTAATGTT
TAATTTATTGCCTTTTAAATTTGCCTCAGTTCATT
CAAAGCTTTCTGAATCTGTTGGATGGATCAACATT
TTGGTTGATTTTATAGAGTATAACCTTCCATTTTG
AGACTTCCAGGATAATCCTCTTCTCCGCTCTGGCT
TAGATTTTGTTCTTGGTTTGTTATATTTACCATTT
AGGTTGTTTTCTCCACACTCATCATGCCACCACCA
GCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTC
CTTTTGCTTTGTGATCCCAAGTAGAAAACACCAAA
TCTTTGTTTTCCGGGATTGCATTGGGGACATTGCC
AGTAATCTTAACTACATGTAGCGTATAGTTGGTTT
CGTGATTTCCCAAGTAAAAAGAATATTCAATATAA
TGTTTGTTGTCTTTCCAGTCTTCCAACTCAATTCG
TAAAACGTAATTAGATTGCTTCACTATGGAGTATA
TCTTCTCTAGGCCCAACCAGAATTCTCCATCAAGC
CTCCCGAAACCATATTTGTAGTTCTCCCACGTTTC
ATTGAAGTTTTGTGATCCATCTATTCTCCTTAGAC
AGGTTTTACCTGATACAACATCACAGTAGACATGA
AAAACTTGAGAGTTGCTGGGTCTGATGGCATACAT
GCCACTTATATGTTCACCTCTATTGTAAATGGTGG
TACAATCAGCAGGAATGCCATCATGTTTTACATTT
CTTATTTCATTCAGCTGAAGAAAGGGAGTAGTTCT
TGGTGCTCTTGGCTTGGAAGATAGAGAAATTTCTG
TGGGTTCTTGAATATTAGTCATTCTGAGCTGATTT
TCTATTTCTTTTATTTGACTGTGCTGTTGGTTTAA
TTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGT
CTTTGATGCTATTATCTTGTTTTTCTACAAAACTT
TTAAGTGAAGTTACTTCTGGATGTTCTGGAGTTTC
AGGTTGATTTTGAATTAAGTTAGTTAGTTGCTCTT
CTAAATATTTCACTTTTTGTTGAAGTAGAATTTTT
TCTTCTAGGAGGCTTTCAAGTTTTGAGTTGAGTTC
AAGTGACATATTCTTTACCTCTTCATTTTTGACTT
GTAGTTTATATGTAGTTCTTCTCAGTTCCTTTTCT
TCTTCTTTGATTTCACTGGTTTGCAGTGATAGATC
ATAAAAAGACTGATCAAATATGTTGAGTTTTTGAA
ATATGTCATTAATTTGGCCCTTAGTCTTATGGACA
AAGTCTTTAAGACCATGTCCCAACTGAAGGAGGCC
ATTGGCTAAAATTTTTACATCGTCTAACATAGCAA
ATCTTGATTTTGGCTCTGGAGATACAGAATCAAAT
GATGAATTGTCTTGGTCAATTCTGGAGGAAATAAC
TAGAGGAACAATAAAAAGAAGGAGCTTAATTGTGA
ACATTTTTATCCTGATTTTCAATTTCAAGCAACGT
GGAACTGTGTTCTTCTGGAAGCAGACCTAGACTTC
TTAACTCTATATAT
Reverse Complement of SEQ ID NO: 3
SEQ ID NO: 7
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAA
TAATTGAGACAAAAAATGCACACAATTAAATTATT
CCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAG
TGGATCCAGACCTTTCATCATTTGATTCTGCACCT
TCAGAGCCAAAATCAAGATTTGCTATGTTGGATGA
TGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGG
GTCATGGACTTAAAGATTTTGTCCATAAGACTAAG
GGACAAATTAACGACATATTTCAGAAGCTCAACAT
ATTTGATCAGTCTTTTTATGACCTATCACTTCGAA
CCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGA
AGAACTACATCTACACTACAAGTTAAAAACGAGGA
GGTGAAGAACATGTCAGTAGAACTGAACTCAAAGC
TTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAA
CACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAA
CTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACC
CAGAAGTAACATCACTCAAAAGTTTTGTAGAACAG
CAAGACAACAGCATAAGAGAACTCCTCCAGAGTGT
GGAAGAACAGTATAAACAATTAAGTCAACAGCACA
TGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAG
ACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTC
TTCTAAATCAAGAGCACCAAGAACTACTCCCCCTC
TTCAACTGAACGAAACAGAAAATACAGAACAAGAT
GACCTTCCTGCCGACTGCTCTGCCGTTTATAACAG
AGGCGAACATACAAGTGGCGTGTACACTATTAAAC
CAAGAAACTCCCAAGGGTTTAATGTCTACTGTGAT
ACCCAATCAGGCAGTCCATGGACATTAATTCAACA
CCGGAAAGATGGCTCACAGGACTTCAACGAAACAT
GGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGAT
GGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGC
TATAGTCCAACAGTCTAACTACATTTTACGACTCG
AGCTACAAGACTGGAAAGACAGCAAGCACTACGTT
GAATACTCCTTTCACCTGGGCAGTCACGAAACCAA
CTACACGCTACATGTGGCTGAGATTGCTGGCAATA
TCCCTGGGGCCCTCCCAGAGCACACAGACCTGATG
TTTTCTACATGGAATCACAGAGCAAAGGGACAGCT
CTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGT
GGAATGACATATGTGGAGAAAACAACCTAAATGGA
AAATACAACAAACCCAGAACCAAATCCAGACCAGA
GAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCA
GAAAGCTCTATGCTATCAAATCATCCAAAATGATG
CTCCAGCCCACCACCTAAGAAGCTTCAACTGAACT
GAGACAAAATAAAAGATCAATAAATTAAATATTAA
AGTCCTCCCGATCACTGTAGTAATCTGGTATTAAA
ATTTTAATGGAAAGCTTGAGAATTGAATTTCAATT
AGGTTTAAACTCATTGTTAAGATCAGATATCACCG
AATCAACGTAAACAAAATTTATC
Reverse Complement of SEQ ID NO: 4
SEQ ID NO: 8
TTTTTTTTTTTTTTTTTTTTTTTTTGATTTTAAGT
ATCTGTTTATTTTTTATTTTTTTACTTATTTTTAT
AGTTTTGTTTTACAATCAAATAGAATTCATATTCT
AGAACATGCAGAGACCCACAGTTATATAGTATATG
TTTATAATATGGAATAATTACAGGTAGGGTTGCAG
GAGTTAGACACCGTATCTCTGACGGTGACCCTGTC
CCTTCTAACTCTCGCTCTGTGTTCCTATCACTTTC
TTTGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCT
GGCCGCGTGTTCCACCACAACTGTGCCTCCAGCCC
ATTTCACCTGTGTTACGTTGTGCTTAGGATATCAC
TGAAAGAGCTCTATACTGCTATCTTTGCAGCTCAT
TTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTA
TACTATGTTCCTTAATAAGACTTATGATAACCATG
AACTATAGAATTAAGTTTCAACAAGCATACTGCAG
GCATGCAGTAGTGATAAGTACAATGAGATCATTGC
AGTTGCTATCATGGATAATATTTACCAATCAGTTT
TATGAAGACTGGGGTCTGCTTTTGCTTATTGTTAC
ATTTCCACAGCCCATTTTATAATTGCTGCTTAATT
TATAATTCTTAATGTCGAGAAGGAGTATCAATGTG
TGGCCCAGACTGGTCTTGAACTTTCAATTCACCTG
CTGCTCCTCCTCCCCCCTTGCCCAAAAGCGCTATG
GTCTCAGGCCTGTGTCAACACACCCAACTAATACT
ATTAAATCAACAGATAAGTGAGTGATGCAAGGAAA
TCACTTTACCATCAAGCCTCCCAAAACCCTTTTCG
TAGTTTTCCCACGTTTGGTTGAAGTTTTGAGAGCC
ATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGC
CTGATTGGGTGTCACAGTAGACATTAAACACTTGA
GAGCTGCTTGGTCTAATAGTATACACGCCACTTGT
ATGTTCACCTCTGTTATAAATGGCAGAGCAGTCAG
CAGGCAGATCATCTTGTTCTATATTTTTTGCTTCC
TTCAGATGAAGAGGGGGAGTAGTTCTTGGTGCTCT
TGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTT
GAATGCCAGTCTTTCTGAGCTGATTTTCTATTTCT
TTTATCTGAATGTGCTGTTGACTTAGTTGTTTATA
TTGTTCTTCCACACTCTGGAGGAGTTCTCTTATGC
TGTTATCTTGCTGTTCTACAAAACTTTTAAGTGAC
GTTACCTCTGGGTGCTCCCGAGCCCCAGGCGGGTT
CTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCC
TGACTCTGTGTTGGAGCGCCATCTTCTCCTCCAGT
AGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACAT
ATTCTTCACCTCTTCGTTTTTAACTTGTAGTTTAG
ATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTG
ATTTCATTGGTTTGAAGTGATAGGTCATAAAAACA
CTGATCAAATATGTTGAGCTTCTGAAATATGTCAT
TAATTTGTCCCTTTGTCTTATGGACAAAATCTTTA
AGACCATGACCCAGCTGCAGGAGGCCATTGGCTAA
AATTTTGACATCATCCAACATAGCAAATCTTGATT
TTGGCTCTGACGGTACAGAATCAAATGGCGAAAGG
TCTGGATCAACTCTGGACGAAATTACTAGAGGAAC
AACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTG
TTTCAATTATTCAATTTCAAGCAATTTGGAACGTC
Macaca fascicularis angiopoietin-
like 3 (Angpt13), mRNA
SEQ ID NO: 9
GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCT
TCCAGAAGAACACAGTTCCACGCTGCTTGAAATTG
AAAATCAGGATAAAAATGTTCACAATTAAGCTCCT
TCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAA
TTGACCAAGACAATTCATCATTTGATTCTGTATCT
CCAGAGCCAAAATCAAGATTTGCTATGTTAGACGA
TGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGG
GACATGGTCTTAAAGACTTTGTCCATAAGACTAAG
GGCCAAATTAATGACATATTTCAAAAACTCAACAT
ATTTGATCAGTCTTTTTATGATCTATCACTGCAAA
CCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGA
AGAACTACATATAAACTACAAGTCAAAAATGAAGA
GGTAAAGAATATGTCACTTGAACTCAACTCAAAAC
TTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAA
CAAAAAGTGAAATATTTAGAAGAGCAACTAACTAA
CTTAATTCAAAATCAACCTGCAACTCCAGAACATC
CAGAAGTAACTTCACTTAAAAGTTTTGTAGAAAAA
CAAGATAATAGCATCAAAGACCTTCTCCAGACTGT
GGAAGAACAATATAAGCAATTAAACCAACAGCATA
GTCAAATAAAAGAAATAGAAAATCAGCTCAGAATG
ACTAATATTCAAGAACCCACAGAAATTTCTCTATC
TTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTC
TTCAGCTGAATGAAATAAGAAATGTAAAACATGAT
GGCATTCCTGCTGATTGTACCACCATTTACAATAG
AGGTGAACATATAAGTGGCACGTATGCCATCAGAC
CCAGCAACTCTCAAGTTTTTCATGTCTACTGTGAT
GTTGTATCAGGTAGTCCATGGACATTAATTCAACA
TCGAATAGATGGATCACAAAACTTCAATGAAACGT
GGGAGAACTACAAATATGGTTTCGGGAGGCTTGAT
GGAGAATTCTGGTTGGGCCTAGAGAAGATATACTC
CATAGTGAAGCAATCTAATTACGTTTTACGAATTG
AGTTGGAAGACTGGAAAGACAACAAACATTATATT
GAATATTCTTTTTACTTGGGAAATCACGAAACCAA
CTATACGCTACATGTAGTTAAGATTACTGGCAATG
TCCCCAATGCAATCCCGGAAAACAAAGATTTGGTG
TTTTCTACTTGGGATCACAAAGCAAAAGGACACTT
CAGCTGTCCAGAGAGTTATTCAGGAGGCTGGTGGT
GGCATGATGAGTGTGGAGAAAACAACCTAAATGGT
AAATATAACAAACCAAGAACAAAATCTAAGCCAGA
GCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATG
GAAGGTTATACTCTATAAAATCAACCAAAATGTTG
ATCCATCCAACAGATTCAGAAAGCTTTGAATGAAC
TGAGGCAAATTTAAAAGGCAATAAATTAAACATTA
AACTCATTCCAAGTTAATGTGGTTTAATAATCTGG
TATTAAATCCTTAAGAGAAGGCTTGAGAAATAGAT
TTTTTTATCTTAAAGTCACTGTCAATTTAAGATTA
AACATACAATCACATAACCTTAAAGAATACCATTT
ACATTTCTCAATCAAAATTCTTACAACACTATTTG
TTTTATATTTTGTGATGTGGGAATCAATTTTAGAT
GGTCGCAATCTAAATTATAATCAACAGGTGAACTT
ACTAAATAACTTTTCTAAATAAAAAACTTAGAGAC
TTTAATTTTAAAAGTCATCATATGAGCTAATGTCA
CAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTT
AAAACTCTAAACTTGACTAAATAAAGAGGACTGAT
AATTATACAGTTCTTAAATTTGTTGTAATATTAAT
TTCAAAACTAAAAATTGTCAGCACAGAGTATGTGT
AAAAATCTGTAATATAAATTTTTAAACTGATGCCT
CATTTTGCTACAAAATAATCTGGAGTAAATTTTTG
ATAGGATTTATTTATGAAACCTAATGAAGCAGGAT
TAAATACTGTATTAAAATAGGTTCGCTGTCTTTTA
AACAAATGGAGATGATGATTACTAAGTCACATTGA
CTTTAATATGAGGTATCACTATACCTTAACATATT
TGTTAAAACGTATACTGTATACATTTTGTGT
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17531885
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alnylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 20th, 2022 03:05PM
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Apr 20th, 2022 03:05PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Apr 19th, 2022 12:00AM
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Nov 22nd, 2021 12:00AM
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https://www.uspto.gov?id=US11306316-20220419
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Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof
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The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.
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11306316
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1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence 5′-AAGACUGAUCAAAUAUGUUGAGU-3′ (SEQ ID NO: 543);
wherein the sense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence 5′-UCAACAUAUUUGAUCAGUCUU-3′ (SEQ ID NO: 358);
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein the sense strand and the antisense strand form a double stranded region of at least 17 nucleotides in length;
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, an abasic nucleotide, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence 5′-AAAGACUGAUCAAAUAUGUUGAG-3′ (SEQ ID NO:1199); wherein the sense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of 5′-CAACAUAUUUGAUCAGUCUUU-3′ (SEQ ID NO:1018);
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein the sense strand and the antisense strand form a double stranded region of at least 17 nucleotides in length,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, an abasic nucleotide, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
3. The dsRNA agent of claim 1, wherein each strand is independently 17-25 nucleotides in length.
4. The dsRNA agent of claim 1, wherein each strand is independently 19-25 nucleotides in length.
5. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
6. The dsRNA of claim 1, wherein the GalNAc (N-acetylgalactosamine) derivative is attached through a bivalent or trivalent branched linker.
7. The dsRNA agent of claim 1, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of
(SEQ ID NO: 387)
5′-CAUAUUUGAUCAGUCUUUUUA-3′
and
(SEQ ID NO: 572)
5′-UAAAAAGACUGAUCAAAUAUGUU-3′;
(SEQ ID NO: 287)
5′-ACAUAUUUGAUCAGUCUUUUU-3′
and
(SEQ ID NO: 472)
5′-AAAAAGACUGAUCAAAUAUGUUG-3′;
(SEQ ID NO: 303)
5′-AACAUAUUUGAUCAGUCUUUU-3′
and
(SEQ ID NO: 488)
5′-AAAAGACUGAUCAAAUAUGUUGA-3′;
(SEQ ID NO: 39)
5′-ACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 101)
5′-AAAGACUGAUCAAAUAUGU-3′;
(SEQ ID NO: 294)
5′-CAACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 479)
5′-AAAGACUGAUCAAAUAUGUUGAG-3′;
(SEQ ID NO: 358)
5′-UCAACAUAUUUGAUCAGUCUU-3′
and
(SEQ ID NO: 543)
5′-AAGACUGAUCAAAUAUGUUGAGU-3′;
and
(SEQ ID NO: 64)
5′-CAACAUAUUUGAUCAGUCU-3′
and
(SEQ ID NO: 126)
5′-AGACUGAUCAAAUAUGUUG-3′.
8. A cell containing the dsRNA agent of claim 1.
9. A pharmaceutical composition for inhibiting expression of an ANGPTL3 gene, comprising the dsRNA agent of claim 1.
10. The pharmaceutical composition of claim 9, wherein the dsRNA agent is present in a buffered solution.
11. A method of inhibiting ANGPTL3 expression in a cell, the method comprising:
(a) contacting the cell with the dsRNA agent of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
12. The method of claim 11, wherein the cell is within a subject.
13. A method of inhibiting the expression of ANGPTL3 in a subject, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby inhibiting the expression of ANGPTL3 in the subject.
14. A method of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating the subject.
15. The method of claim 14, wherein the disorder is a disorder of lipid metabolism.
16. The method of claim 14, wherein the disorder is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, atherosclerosis, diabetes, cardiovascular disease, and coronary artery disease.
17. The method of claim 14, further comprising administering an additional therapeutic to the subject.
18. The method of claim 17, wherein the additional therapeutic is a statin.
19. The method of claim 14, wherein the dsRNA agent is administered at a dose of about 0.5 mg/kg to about 50 mg/kg.
20. The method of claim 14, wherein the administration of the dsRNA agent to the subject causes a decrease in one or more serum lipid and/or a decrease in ANGPTL3 protein accumulation.
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20
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RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 17/089,854, filed on Nov. 5, 2020, which is a continuation of U.S. patent application Ser. No. 16/411,261, filed on May 14, 2019, now, U.S. Pat. No. 10,934,545, issued on Mar. 2, 2021, which is a continuation of U.S. patent application Ser. No. 15/683,999, filed on Aug. 23, 2017, now U.S. Pat. No. 10,337,010, issued on Jul. 2, 2019, which is a continuation of U.S. patent application Ser. No. 15/068,912 filed on Mar. 14, 2016, now U.S. Pat. No. 9,771,591, issued on Sep. 26, 2017, which is a continuation of U.S. patent application Ser. No. 14/132,999 filed on Dec. 18, 2013, now U.S. Pat. No. 9,322,018, issued on Apr. 26, 2016, which is a 35 U.S.C. 111(a) continuation application, which claims priority to PCT/US2012/043378, filed on Jun. 20, 2012, U.S. Provisional Application No. 61/499,620, filed on Jun. 21, 2011, and to U.S. Provisional Application No. 61/638,288, filed on Apr. 25, 2012. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 19, 2021, is named 121301_00312_SL.txt and is 444,512 bytes in size.
BACKGROUND OF THE INVENTION
Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulates lipid metabolism and that is predominantly expressed in the liver (Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activities of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and of endothelial lipase (EL), which hydrolyzes high density lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese, KK/Snk mice, a reduction in ANGPTL3 expression has a protective effect against hyperlipidemia and artherosclerosis by promoting the clearance of triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223). Human ANGPTL3 plasma concentrations positively correlate with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).
Disorders of lipid metabolism can lead to elevated levels of serum lipids, such as triglycerides and/or cholesterol. Elevated serum lipids are strongly associated with high blood pressure, cardiovascular disease, diabetes and other pathologic conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder that is characterized by high blood levels of triglycerides. It has been associated with atherosclerosis, even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia can also lead to pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder that is characterized by elevated levels of any one or all lipids and/or lipoproteins in the blood. Current treatments for disorders of lipid metabolism, including dieting, exercise and treatment with statins and other drugs, are not always effective. Accordingly, there is a need in the art for alternative treatments for subjects having disorders of lipid metabolism.
SUMMARY OF THE INVENTION
The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPL3 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ANGPL3 gene, e.g., a subject suffering or prone to suffering from a disorder of lipid metabolism, such as a subject suffering or prone to suffering from hyperlipidemia or hypertriglyceridemia.
Accordingly, in one aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.
In another aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.
In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of AD-53063.1, AD-53001.1, AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 of Tables 7 and 8.
In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length. In one embodiment, each strand of a dsRNA is no more than 30 nucleotides in length.
At least one strand of a dsRNA may comprise a 3′ overhang of at least 1 nucleotide or at least 2 nucleotides.
In certain embodiments, a dsRNA further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
In some embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is
In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic
In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.
In one embodiment, the region of complementarity of a dsRNA consists of one of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, a dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10.
In another aspect, the present invention provides a cell, e.g., a hepatocyte, containing a dsRNA of the invention.
In yet another aspect, the present invention provides a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage. The region of complementarity may be least 15 nucleotides in length or 19 to 21 nucleotides in length.
In a further aspect, the present invention provides a cell comprising a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.
In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising a dsRNA or vector of the invention.
In one embodiment, the pharmaceutical composition comprises a lipid formulation, such as a MC3, SNALP or XTC formulation.
In another aspect, the present invention provides methods of inhibiting ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA or a vector of the invention, and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
The cell may be within a subject, such as a human subject, for example a human subject suffering from a disorder of lipid metabolism, e.g., hyperlipidemia or hypertriglyceridemia.
In one embodiment of the methods of the invention, ANGPTL3 expression is inhibited by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby treating the subject.
The disorder may be disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia
In one embodiment, the administration of the dsRNA to the subject causes a decrease in the level of a serum lipid, triglycerides, cholesterol and/or free fatty acids; and/or a decrease in ANGPTL3 protein accumulation. In one embodiment, administration of the dsRNA to the subject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, IDL-C and/or total cholesterol.
In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another aspect, the present invention provides methods of inhibiting the expression of ANGPTL3 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby inhibiting the expression of ANGPTL3 in the subject.
In yet another aspect, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method of inhibiting expression of ANGPTL3 gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the ANGPTL3 in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of the experimental procedure used for in vivo tests described in Example 2.
FIG. 2A is a graph showing measured levels of ANGPTL3 protein in WT mice after treatment with the indicated iRNA or a control.
FIG. 2B is a graph showing measured levels of ANGPTL3 protein in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 3A is a graph showing measured levels of LDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 3B is a graph showing measured levels of LDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 4A is a graph showing measured levels of triglycerides in WT mice after treatment with the indicated iRNA or a control.
FIG. 4B is a graph showing measured levels of triglycerides in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 5A is a graph showing measured levels of total cholesterol (TC) in WT mice after treatment with the indicated iRNA or a control.
FIG. 5B is a graph showing measured levels of total cholesterol (TC) in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 6A is a graph showing measured levels of HDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 6B is a graph showing measured levels of HDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 7 is a graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or a control.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPTL3gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ANGPTL3 gene. The use of these iRNAs enables the targeted degradation of mRNAs of an ANGPTL3 gene in mammals. Very low dosages of ANGPTL3 iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of an ANGPTL3 gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of expression of an ANGPTL3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of an ANGPTL3 protein, such as a subject having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ANGPTL3 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.
I. Definitions
In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.
The term “ANGPTL3” refers to an angiopoietin like protein 3 having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3. The term encompasses full-length unprocessed precursor forms of ANGPTL3 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the fibrinogen-like domain. The sequence of a human ANGPTL3 mRNA transcript can be found at, for example, GenBank Accession No. GI: 41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesus ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 142388354 (NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 68163568 (NM_001025065.1; SEQ ID NO:4).
The term“ANGPTL3” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the ANGPTL3 gene, such as a single nucleotide polymorphism in the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the ANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039; rs192778191; rs192764027; rs192528948; rs191931953; rs191293319; rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3gene.
The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of ANGPTL3 in a cell, e.g., a cell within a subject, such as a mammalian subject.
In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an ANGPTL3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.
In another aspect, the RNAi agent is a single-stranded antisense RNA molecule. An antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule may be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an ANGPTL3 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.
As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an ANGPTL3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.
The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a part of an ANGPTL3mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ANGPTL3.
In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.
The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of an ANGPTL3,” as used herein, includes inhibition of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 gene that encode an ANGPTL3 protein.
“Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
The expression of an ANGPTL3 gene may be assessed based on the level of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of an ANGPTL3 may also be assessed indirectly based on the levels of a serum lipid, a triglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
In one embodiment, at least partial suppression of the expression of an ANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNA which can be isolated from or detected in a first cell or group of cells in which an ANGPTL3 gene is transcribed and which has or have been treated such that the expression of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:
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(
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100
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The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.
In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.
The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817, the entire contents of which are hereby incorporated herein by reference. Examples of “SNALP” formulations are described below.
As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human having a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as lowering levels of triglycerides in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a disorder of lipid metabolism, such as, e.g., a decrease in the size of eruptive xanthomas. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., high triglyceride levels or eruptive xanthoma. The likelihood of developing a high tryglyceride levels or eruptive xanthoma is reduced, for example, when an individual having one or more risk factors for a high tryglyceride levels or eruptive xanthoma either fails to develop high tryglyceride levels or eruptive xanthoma or develops high tryglyceride levels or eruptive xanthoma with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition i (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.
As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e.g., a lipoprotein complex. Non-limiting examples of serum lipids may include triglycerides and cholesterol, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).
As used herein, a “disorder of lipid metabolism” refers to any disorder associated with or caused by a disturbance in lipid metabolism. For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, or an acquired disorder, such as a disorder acquired as a result of a diet or intake of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrom, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).
Cardiovascular diseases associated with disorders of lipid metabolism are also considered “disorders of lipid metabolism”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke.
Disorders related to body weight are also considered “disorders of lipid metabolism”, as defined herein. Such disorders may include obesity, metabolic syndrome including independent components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss.
Blood sugar disorders are further considered “disorders of lipid metabolism”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of lipid metabolism may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.
“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a disorder of lipid metabolism, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a disorder of lipid metabolism, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.
II. iRNAs of the Invention
Described herein are iRNAs which inhibit the expression of an ANGPTL3 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a disorder of lipid metabolism, e.g., familial hyperlipidemia. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ANGPTL3gene, The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, a primate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.
A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an ANGPTL3gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ANGPTL3 expression is not generated in the target cell by cleavage of a larger dsRNA.
A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.
In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in Tables 2, 3, 7, 8, 9 and 10, and the corresponding antisense strand of the sense strand is selected from the group of sequences of Tables 2, 3, 7, 8, 9 and 10. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an ANGPTL3gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in Tables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.
The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 7, 8, 9 and 10, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2, 3, 7, 8, 9 and 10 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 2, 3, 7, 8, 9 and 10, and differing in their ability to inhibit the expression of an ANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.
In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify a site(s) in an ANGPTL3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 7, 8, 9 and 10 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ANGPTL3gene.
While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and 10 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 7, 8, 9 and 10, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.
An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an ANGPTL3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ANGPTL3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ANGPTL3 gene is important, especially if the particular region of complementarity in an ANGPTL3 gene is known to have polymorphic sequence variation within the population.
III. Modified iRNAs of the Invention
In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.
Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.
IV. iRNAs Conjugated to Ligands
Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.
Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
A. Lipid Conjugates
In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).
B. Cell Permeation Agents
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
C. Carbohydrate Conjugates
In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as
In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:
Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,
when one of X or Y is an oligonucleotide, the other is a hydrogen.
In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
D. Linkers
In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.
The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
i. Redox Cleavable Linking Groups
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
ii. Phosphate-Based Cleavable Linking Groups
In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.
iii. Acid Cleavable Linking Groups
In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
iv. Ester-Based Linking Groups
In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
v. Peptide-Based Cleaving Groups
In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,
when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):
wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
p2A, p2B, p3A, p3B, p4A, p4B, p5A, p5B, p5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O);
R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO,
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):
wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II_VII, XI, X, and XIII.
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.
“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
IV. Delivery of an iRNA of the Invention
The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et a.l (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
A. Vector Encoded iRNAs of the Invention
iRNA targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., (1993) Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood 83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; and Wang et al., (1995) Gene Therapy 2:775-783. A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
V. Pharmaceutical Compositions of the Invention
The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, such as hypertriglyceridemia.
Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.
The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a ANGPTL3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on ANGPTL3 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of lipid metabolism that would benefit from reduction in the expression of ANGPTL3. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of an LDL receptor (LDLR−/− mouse; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced artherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).
The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
A. iRNA Formulations Comprising Membranous Molecular Assemblies
An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.
A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.
Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).
Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S. T. P. Pharma. Sci., 4(6):466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.
Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.
Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
B. Nucleic Acid Lipid Particles
iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.
The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary lipid-dsRNA formulations are described in the table below.
cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid
Ionizable/Cationic Lipid
Lipid:siRNA ratio
conjugate
SNALP-1
12-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-cDMA
dimethylaminopropane(DLmDMA)
(57.1/7.1/34.4/1.4)
lipid:siRNA~7:1
2-XTC
2,2-Dilinoleyl-4-dimethylaminoethy-
XTC/DPPC/Cholesterol/PEG-cDMA
[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4
lipid:siRNA~7:1
LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA~6:1
LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA~11:1
LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA~6:1
LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA~11:1
LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
50/10/38.5/1.5
Lipid:siRNA 10:1
LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG
di((9Z,12Z)-octadeca-9,12-dienyl)
50/10/38.5/1.5
tetrahydro-3aH-cyclopenta[d][1,3]
Lipid:siRNA 10:1
dioxol-5-amine (ALN100)
LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG
6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5
(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1
LNP12
1,1′-(2-(4-(2((2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-DMG
hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5
hydroxydodecyl)amino)ethyl)
Lipid:siRNA 10:1
piperazin-1-yl)ethylazanediyl)
didodecan-2-ol (Tech G1)
LNP13
XTC
XTC/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 33:1
LNP14
MC3
MC3/DSPC/Chol/PEG-DMG
40/15/40/5
Lipid:siRNA: 11:1
LNP15
MC3
MC3/DSPC/Chol/PEG-
DSG/Ga1NAc-PEG-DSG
50/10/35/4.5/0.5
Lipid:siRNA: 11:1
LNP16
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP17
MC3
MC3/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP18
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 12:1
LNP19
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/35/5
Lipid:siRNA: 8:1
LNP20
MC3
MC3/DSPC/Chol/PEG-DPG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP22
XTC
XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
DSPC: distearoylphosphatidylcholine
DPPC: dipalmitoylphosphatidylcholine
PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.
ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
Synthesis of Ionizable/Cationic Lipids
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
Synthesis of 515
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H] −232.3 (96.94%).
Synthesis of 517A and 517B
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an. Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR δ=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
C. Additional Formulations
i. Emulsions
The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
ii. Microemulsions
In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
iii. Microparticles
an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
iv. Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.
Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
v. Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′ isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
vi. Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
vii. Other Components
The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a disorder of lipid metabolism. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by ANGPTL3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
VI. Methods of the Invention
The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression of ANGPTL3 may also be assessed indirectly by measuring a decrease in biological activity of ANGPTL3, e.g., a decrease in the level of serum lipid, triglycerides, cholesterol and/or free fatty acids.
In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
A cell suitable for treatment using the methods of the invention may be any cell that expresses an ANGPTL3gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.
ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.
The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ANGPTL3 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.
In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.
In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.
The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
In one aspect, the present invention also provides methods for inhibiting the expression of an ANGPTL3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an ANGPTL3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in ANGPTL3 gene and/or protein expression.
The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of ANGPTL3 expression, in a therapeutically effective amount of an iRNA targeting an ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting an ANGPTL3 gene.
An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.
Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
Subjects that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression are those having a disorder of lipid metabolism, e.g., an inherited disorder of lipid metabolism or an acquired disorder of lipid metabolism. In one embodiment, a subject having disorder of lipid metabolism has hyperlipidemia. In another embodiment, a subject having a disorder of lipid metabolism has hypertriglyceridemia. Treatment of a subject that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., a subject is having eruptive xanthomas) and prophylactic treatment (e.g., the subject is not having eruptive xanthomas or a subject may be at risk of developing eruptive xanthomas).
The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting ANGPTL3 is administered in combination with, e.g., an agent useful in treating a disorder of lipid metabolism as described elsewhere herein. For example, additional agents suitable for treating a subject that would benefit from reducton in ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, may include agents that lower one or more serum lipids. Non-limiting examples of such agents may include cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors, e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). Other agents useful in treating a disorder of lipid metabolism may include bile sequestering agents, such as cholestyramine and other resins; VLDL secretion inhibitors, such as niacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterol acyl transferase inhibitors; farnesoid X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptide; and therapeutic antibodies against ANGPTL3. The additional therapeutic agents may also include agents that raise high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, the additional therapeutic agents may also include dietary supplements, e.g., fish oil. The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.
In one embodiment, the method includes administering a composition featured herein such that expression of the target ANGPTL3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target ANGPTL3 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.
Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target ANGPTL3gene.
Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein. Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ANGPTL3 or pharmaceutical composition thereof, “effective against” a disorder of lipid metabolism indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating disorder of lipid metabolisms and the related causes.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.
Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of dsRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of dsRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.
Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 days. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some embodiments, the dosing may comprise a loading phase of multiple doses on consecutive days.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Transcripts siRNA design was carried out to identify siRNAs targeting the human ANGPTL3 transcript annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/) and a cynomolgus monkey (Macaca fascicularis; henceforth “cyno”) ANGPTL3 transcript produced via sequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was done in-house, and the mRNA sequence is shown in SEQ ID NO:9. Design used the following transcripts from the NCBI collection: Human—NM_014495.2 (SEQ ID NO:1); Mouse—NM_013913.3 (SEQ ID NO:2). All siRNA duplexes were designed that shared 100% identity with the listed human and cyno transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in NCBI Gene database.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 977 candidate human/cyno siRNAs, and a subset of 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) were then used in a comprehensive search against the human transcriptome (defined as the set of NM_ and XM_records within the human NCBI Refseq set) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start were used to create 2 heptamers and one octomer. ‘Heptamer1’ was created by adding a 3′ A to the hexamer; ‘heptamer2’ was created by adding a 5′ A to the hexamer; octomer was created by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3×normalized octomer count)+(2×heptamer2 count)+(1×heptamer1 count)).
Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. Sorting was carried out by the specificity of the antisense strand. Duplexes were then selected from the human/cyno set with antisense oligos lacking miRNA seed matches, scores of 3 or better, less than 65% overall GC content, no GC at the first position, 4 or more Us or As in the seed region, and GC at the nineteenth position. Duplexes from the human/cyno/mouse set with antisense oligos having scores of 2 or better, less than 65% overall GC content, and no GC at the first position were also selected.
siRNA Sequence Selection
A total of 47 sense and 47 antisense derived siRNA oligos from the human/cyno set were synthesized and formed into duplexes. A total of 15 sense and 15 antisense derived siRNAs from the human/cyno/mouse set were synthesized and formed into duplexes.
Synthesis of ANGPTL3 Sequences
ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either a 1 or 0.2 μmol scale. Single strands were synthesized with 2′O-methyl modifications for transfection based in vitro screening. For use in free uptake screening assays, 3′ GalNAc conjugates were made with 2′ F and 2′-O-methyl chemical modifications. In these designs, GalNAc moiety was placed at the 3′ end of the sense strand. The antisense sequence was 23 nucleotides in length and also contained 2′ F and 2′ Omethyl chemical modifications with two phosphorothioate linkages at the 3′ end.
On one set of 21mer single strands and duplexes, ‘endolight’ chemistry was applied as detailed below.
All pyrimidines (cytosine and uridine) in the sense strand were modified with 2′-O-Methyl nucleotides (2′ O-Methyl C and 2′-O-Methyl U)
In the antisense strand, pyrimidines adjacent (towards 5′ position) to ribo A nucleoside were replaced with their corresponding 2′-O-Methyl nucleosides
A two base dTsdT extension at the 3′ end of both sense and anti sense sequences was introduced
For GalNAc conjugated 21mer sense and complementary 23mer antisense sequences, 2′ F and 2′ OMethyl modified single strands were synthesized. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support for the antisense sequence at a 1 μmol scale. The sequence motif named TOFFEE was applied, in which the sense strand contained a three-nucleotide 2′ F-modified motif at positions 9, 10 and 11 and in the antisense, a 2′ OMethyl-modified motif was included at positions 11, 12 and 13.
Synthesis, Cleavage and Deprotection
The synthesis of ANGPTL3 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for the sense strand and a universal CPG for the antisesense strand were used.
The synthesis of the above sequences was performed at either a 1 or 0.2 μm scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as the activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′ F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mix and the pellets were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification, Desalting and Annealing
ANGPTL3 sequences were precipitated and purified on an AKTA Purifier system using a Sephadex column. The ANGPTL3 was run at ambient temperature. Sample injection and collection was performed in 96 well plates with 1.8 mL deep wells. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ANGPTL3 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.
Example 2. In Vitro Screening
Cell Culture and Transfections
Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×104 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.
Free Uptake Transfection
5 μl of each GalNac conjugated siRNA in PBS was combined with 4×104 freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspended in 95 μl of In Vitro Gro CP media (In Vitro Technologies-Celsis, Baltimore, Md.) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37° C. in an atmosphere of 5% CO2. siRNAs were tested at final concentrations of 500 nM, 100 nM and 10 nM for efficacy free uptake assays. For dose response screens, final siRNA concentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl of Wash Buffer B, captured, and the supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured, and the supernatant was removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 2 μl 10×Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR
2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat #Hs00205581_ml) and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).
Viability Screens
Cell viability was measured on days 3 and 6 in HeLa and Hep3B cells following transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells were plated at a density of 10,000 cells per well in 96 well plates. Each siRNA was assayed in triplicate and the data averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability, and AD-1955 and mock transfected cells as negative controls. PLK1 and AD-19200 result in a dose dependent loss of viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96 well plates after 3 or 6 days and incubated at 37° C. for 2 hours. Plates were then read in a Spectrophotometer (Molecular Devices) at 560Ex/590Em. Viability was expressed as the average value of light units from three replicate transfections+/−standard deviation. Relative viability was assessed by first averaging the three replicate transfections and then normalizing Mock transfected cells. Data is expressed as % viabile cells.
TABLE 1
Abbreviations of nucleotide monomers used in nucleic
acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are
mutually linked by 5′-3′-phosphodiester bonds.
Abbreviation
Nucleotide(s)
A
adenosine
C
cytidine
G
guanosine
T
thymidine
U
uridine
N
any nucleotide (G, A, C, T or U)
a
2′-O-methyladenosine
c
2′-O-methylcytidine
g
2′-O-methylguanosine
u
2′-O-methyluridine
dT
2′-deoxythymidine
s
phosphorothioate linkage
TABLE 2
Unmodified sense and antisense strand sequences of ANGPTL3 dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 16-77,
(SEQ ID NOS 78-139,
Sense
respectively,
Position in
Antisense
respectively,
Position in
Duplex ID
Name
in order of appearance)
NM_014495.2
Name
in order of appearance)
NM_014495.2
AD-45939.1
A-96225.1
UAUUUGAUCAGUCUUUUUA
281-299
A-96226.1
UAAAAAGACUGAUCAAAUA
281-299
AD-45858.1
A-96149.1
GAGCAACUAACUAACUUAA
478-496
A-96150.1
UUAAGUUAGUUAGUUGCUC
478-496
AD-45869.1
A-96137.1
GGCCAAAUUAAUGACAUAU
247-265
A-96138.1
AUAUGUCAUUAAUUUGGCC
247-265
AD-45884.1
A-96189.1
CGAAUUGAGUUGGAAGACU
1045-1063
A-96190.1
AGUCUUCCAACUCAAUUCG
1045-1063
AD-45892.1
A-96129.1
CCUCCUUCAGUUGGGACAU
198-216
A-96130.1
AUGUCCCAACUGAAGGAGG
198-216
AD-45899.1
A-96147.1
CACUUGAACUCAACUCAAA
401-419
A-96148.1
UUUGAGUUGAGUUCAAGUG
401-419
AD-45915.1
A-96231.1
GUCCAUGGACAUUAAUUCA
890-908
A-96232.1
UGAAUUAAUGUCCAUGGAC
890-908
AD-45924.1
A-96219.1
AAUCAAGAUUUGCUAUGUU
152-170
A-96220.1
AACAUAGCAAAUCUUGAUU
152-170
AD-45860.1
A-96181.1
CUAGAGAAGAUAUACUCCA
1000-1018
A-96182.1
UGGAGUAUAUCUUCUCUAG
1000-1018
AD-45870.1
A-96153.1
CUAACUAACUUAAUUCAAA
484-502
A-96154.1
UUUGAAUUAAGUUAGUUAG
484-502
AD-45870.2
A-96153.2
CUAACUAACUUAAUUCAAA
484-502
A-96154.2
UUUGAAUUAAGUUAGUUAG
484-502
AD-45877.1
A-96171.1
CAUUAAUUCAACAUCGAAU
899-917
A-96172.1
AUUCGAUGUUGAAUUAAUG
899-917
AD-45885.1
A-96205.1
CAAAAUGUUGAUCCAUCCA
1392-1410
A-96206.1
UGGAUGGAUCAACAUUUUG
1392-1410
AD-45893.1
A-96145.1
CAUAUAAACUACAAGUCAA
359-377
A-96146.1
UUGACUUGUAGUUUAUAUG
359-377
AD-45900.1
A-96163.1
GACCCAGCAACUCUCAAGU
839-857
A-96164.1
ACUUGAGAGUUGCUGGGUC
839-857
AD-45925.1
A-96235.1
GGUUGGGCCUAGAGAAGAU
992-1010
A-96236.1
AUCUUCUCUAGGCCCAACC
992-1010
AD-45861.1
A-96197.1
GUGUGGAGAAAACAACCUA
1272-1290
A-96198.1
UAGGUUGUUUUCUCCACAC
1272-1290
AD-45871.1
A-96169.1
GACAUUAAUUCAACAUCGA
897-915
A-96170.1
UCGAUGUUGAAUUAAUGUC
897-915
AD-45878.1
A-96187.1
CAUAGUGAAGCAAUCUAAU
1017-1035
A-96188.1
AUUAGAUUGCUUCACUAUG
1017-1035
AD-45886.1
A-96127.1
CUAUGUUAGACGAUGUAAA
164-182
A-96128.1
UUUACAUCGUCUAACAUAG
164-182
AD-45894.1
A-96161.1
CACAGAAAUUUCUCUAUCU
684-702
A-96162.1
AGAUAGAGAAAUUUCUGUG
684-702
AD-45901.1
A-96179.1
GUUGGGCCUAGAGAAGAUA
993-1011
A-96180.1
UAUCUUCUCUAGGCCCAAC
993-1011
AD-45909.1
A-96213.1
GCCAAAAUCAAGAUUUGCU
147-165
A-96214.1
AGCAAAUCUUGAUUUUGGC
147-165
AD-45934.1
A-96223.1
ACAUAUUUGAUCAGUCUUU
278-296
A-96224.1
AAAGACUGAUCAAAUAUGU
278-296
AD-45934.2
A-96223.2
ACAUAUUUGAUCAGUCUUU
278-296
A-96224.2
AAAGACUGAUCAAAUAUGU
278-296
AD-45863.1
A-96135.1
CUUAAAGACUUUGUCCAUA
220-238
A-96136.1
UAUGGACAAAGUCUUUAAG
220-238
AD-45872.1
A-96185.1
CCAUAGUGAAGCAAUCUAA
1016-1034
A-96186.1
UUAGAUUGCUUCACUAUGG
1016-1034
AD-45879.1
A-96203.1
CAACCAAAAUGUUGAUCCA
1388-1406
A-96204.1
UGGAUCAACAUUUUGGUUG
1388-1406
AD-45887.1
A-96143.1
CUACAUAUAAACUACAAGU
356-374
A-96144.1
ACUUGUAGUUUAUAUGUAG
356-374
AD-45895.1
A-96177.1
GGGAGGCUUGAUGGAGAAU
970-988
A-96178.1
AUUCUCCAUCAAGCCUCCC
970-988
AD-45902.1
A-96195.1
GGUGUUUUCUACUUGGGAU
1188-1206
A-96196.1
AUCCCAAGUAGAAAACACC
1188-1206
AD-45910.1
A-96229.1
AAGAGCACCAAGAACUACU
711-729
A-96230.1
AGUAGUUCUUGGUGCUCUU
711-729
AD-45935.1
A-96239.1
UGGAGAAAACAACCUAAAU
1275-1293
A-96240.1
AUUUAGGUUGUUUUCUCCA
1275-1293
AD-45864.1
A-96151.1
GCAACUAACUAACUUAAUU
480-498
A-96152.1
AAUUAAGUUAGUUAGUUGC
480-498
AD-45873.1
A-96201.1
CAACCUAAAUGGUAAAUAU
1284-1302
A-96202.1
AUAUUUACCAUUUAGGUUG
1284-1302
AD-45880.1
A-96125.1
GCUAUGUUAGACGAUGUAA
163-181
A-96126.1
UUACAUCGUCUAACAUAGC
163-181
AD-45888.1
A-96159.1
CCCACAGAAAUUUCUCUAU
682-700
A-96160.1
AUAGAGAAAUUUCUGUGGG
682-700
AD-45896.1
A-96193.1
GAUUUGGUGUUUUCUACUU
1183-1201
A-96194.1
AAGUAGAAAACACCAAAUC
1183-1201
AD-45903.1
A-96211.1
CAGAGCCAAAAUCAAGAUU
143-161
A-96212.1
AAUCUUGAUUUUGGCUCUG
143-161
AD-45919.1
A-96217.1
AAAUCAAGAUUUGCUAUGU
151-169
A-96218.1
ACAUAGCAAAUCUUGAUUU
151-169
AD-45865.1
A-96167.1
CAUGGACAUUAAUUCAACA
893-911
A-96168.1
UGUUGAAUUAAUGUCCAUG
893-911
AD-45874.1
A-96123.1
GAUUUGCUAUGUUAGACGA
158-176
A-96124.1
UCGUCUAACAUAGCAAAUC
158-176
AD-45881.1
A-96141.1
GAACUACAUAUAAACUACA
353-371
A-96142.1
UGUAGUUUAUAUGUAGUUC
353-371
AD-45889.1
A-96175.1
CGAAUAGAUGGAUCACAAA
913-931
A-96176.1
UUUGUGAUCCAUCUAUUCG
913-931
AD-45897.1
A-96209.1
CUUGUUAAAACUCUAAACU
1817-1835
A-96210.1
AGUUUAGAGUUUUAACAAG
1817-1835
AD-45904.1
A-96227.1
AUUUGAUCAGUCUUUUUAU
282-300
A-96228.1
AUAAAAAGACUGAUCAAAU
282-300
AD-45920.1
A-96233.1
UCCAUGGACAUUAAUUCAA
891-909
A-96234.1
UUGAAUUAAUGUCCAUGGA
891-909
AD-45856.1
A-96117.1
CACAAUUAAGCUCCUUCUU
57-75
A-96118.1
AAGAAGGAGCUUAAUUGUG
57-75
AD-45929.1
A-96221.1
CAACAUAUUUGAUCAGUCU
276-294
A-96222.1
AGACUGAUCAAAUAUGUUG
276-294
AD-45866.1
A-96183.1
CUCCAUAGUGAAGCAAUCU
1014-1032
A-96184.1
AGAUUGCUUCACUAUGGAG
1014-1032
AD-45875.1
A-96139.1
GCCAAAUUAAUGACAUAUU
248-266
A-96140.1
AAUAUGUCAUUAAUUUGGC
248-266
AD-45882.1
A-96157.1
CAACAGCAUAGUCAAAUAA
622-640
A-96158.1
UUAUUUGACUAUGCUGUUG
622-640
AD-45890.1
A-96191.1
GGAAAUCACGAAACCAACU
1105-1123
A-96192.1
AGUUGGUUUCGUGAUUUCC
1105-1123
AD-45898.1
A-96131.1
CAGUUGGGACAUGGUCUUA
205-223
A-96132.1
UAAGACCAUGUCCCAACUG
205-223
AD-45857.1
A-96133.1
GACAUGGUCUUAAAGACUU
212-230
A-96134.1
AAGUCUUUAAGACCAUGUC
212-230
AD-45930.1
A-96237.1
UGUGGAGAAAACAACCUAA
1273-1291
A-96238.1
UUAGGUUGUUUUCUCCACA
1273-1291
AD-45867.1
A-96199.1
GUGGAGAAAACAACCUAAA
1274-1292
A-96200.1
UUUAGGUUGUUUUCUCCAC
1274-1292
AD-45876.1
A-96155.1
CCAACAGCAUAGUCAAAUA
621-639
A-96156.1
UAUUUGACUAUGCUGUUGG
621-639
AD-45883.1
A-96173.1
CAACAUCGAAUAGAUGGAU
907-925
A-96174.1
AUCCAUCUAUUCGAUGUUG
907-925
AD-45891.1
A-96207.1
GCAAAUUUAAAAGGCAAUA
1441-1459
A-96208.1
UAUUGCCUUUUAAAUUUGC
1441-1459
AD-45914.1
A-96215.1
CAAAAUCAAGAUUUGCUAU
149-167
A-96216.1
AUAGCAAAUCUUGAUUUUG
149-167
AD-15838.1
A-26242.1
AGAGCCAAAAUCAAGAUUU
144-162
A-26243.2
AAAUCUUGAUUUUGGCUCU
144-162
TABLE 3
Modified sense and antisense strand sequences of ANGPTL3 dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 140-201,
(SEQ ID NOS 202-263,
Sense
respectively,
Antisense
respectively,
Duplex ID
OligoName
in order of appearance)
OligoName
in order of appearance)
AD-45939.1
A-96225.1
uAuuuGAucAGucuuuuuAdTsdT
A-96226.1
uAAAAAGACUGAUcAAAuAdTsdT
AD-45858.1
A-96149.1
GAGcAAcuAAcuAAcuuAAdTsdT
A-96150.1
UuAAGUuAGUuAGUUGCUCdTsdT
AD-45869.1
A-96137.1
GGccAAAuuAAuGAcAuAudTsdT
A-96138.1
AuAUGUcAUuAAUUUGGCCdTsdT
AD-45884.1
A-96189.1
cGAAuuGAGuuGGAAGAcudTsdT
A-96190.1
AGUCUUCcAACUcAAUUCGdTsdT
AD-45892.1
A-96129.1
ccuccuucAGuuGGGAcAudTsdT
A-96130.1
AUGUCCcAACUGAAGGAGGdTsdT
AD-45899.1
A-96147.1
cAcuuGAAcucAAcucAAAdTsdT
A-96148.1
UUUGAGUUGAGUUcAAGUGdTsdT
AD-45915.1
A-96231.1
GuccAuGGAcAuuAAuucAdTsdT
A-96232.1
UGAAUuAAUGUCcAUGGACdTsdT
AD-45924.1
A-96219.1
AAucAAGAuuuGcuAuGuudTsdT
A-96220.1
AAcAuAGcAAAUCUUGAUUdTsdT
AD-45860.1
A-96181.1
cuAGAGAAGAuAuAcuccAdTsdT
A-96182.1
UGGAGuAuAUCUUCUCuAGdTsdT
AD-45870.1
A-96153.1
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.1
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45870.2
A-96153.2
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.2
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45877.1
A-96171.1
cAuuAAuucAAcAucGAAudTsdT
A-96172.1
AUUCGAUGUUGAAUuAAUGdTsdT
AD-45885.1
A-96205.1
cAAAAuGuuGAuccAuccAdTsdT
A-96206.1
UGGAUGGAUcAAcAUUUUGdTsdT
AD-45893.1
A-96145.1
cAuAuAAAcuAcAAGucAAdTsdT
A-96146.1
UUGACUUGuAGUUuAuAUGdTsdT
AD-45900.1
A-96163.1
GAcccAGcAAcucucAAGudTsdT
A-96164.1
ACUUGAGAGUUGCUGGGUCdTsdT
AD-45925.1
A-96235.1
GGuuGGGccuAGAGAAGAudTsdT
A-96236.1
AUCUUCUCuAGGCCcAACCdTsdT
AD-45861.1
A-96197.1
GuGuGGAGAAAAcAAccuAdTsdT
A-96198.1
uAGGUUGUUUUCUCcAcACdTsdT
AD-45871.1
A-96169.1
GAcAuuAAuucAAcAucGAdTsdT
A-96170.1
UCGAUGUUGAAUuAAUGUCdTsdT
AD-45878.1
A-96187.1
cAuAGuGAAGcAAucuAAudTsdT
A-96188.1
AUuAGAUUGCUUcACuAUGdTsdT
AD-45886.1
A-96127.1
cuAuGuuAGAcGAuGuAAAdTsdT
A-96128.1
UUuAcAUCGUCuAAcAuAGdTsdT
AD-45894.1
A-96161.1
cAcAGAAAuuucucuAucudTsdT
A-96162.1
AGAuAGAGAAAUUUCUGUGdTsdT
AD-45901.1
A-96179.1
GuuGGGccuAGAGAAGAuAdTsdT
A-96180.1
uAUCUUCUCuAGGCCcAACdTsdT
AD-45909.1
A-96213.1
GccAAAAucAAGAuuuGcudTsdT
A-96214.1
AGcAAAUCUUGAUUUUGGCdTsdT
AD-45934.1
A-96223.1
AcAuAuuuGAucAGucuuudTsdT
A-96224.1
AAAGACUGAUcAAAuAUGUdTsdT
AD-45934.2
A-96223.2
AcAuAuuuGAucAGucuuudTsdT
A-96224.2
AAAGACUGAUcAAAuAUGUdTsdT
AD-45863.1
A-96135.1
cuuAAAGAcuuuGuccAuAdTsdT
A-96136.1
uAUGGAcAAAGUCUUuAAGdTsdT
AD-45872.1
A-96185.1
ccAuAGuGAAGcAAucuAAdTsdT
A-96186.1
UuAGAUUGCUUcACuAUGGdTsdT
AD-45879.1
A-96203.1
cAAccAAAAuGuuGAuccAdTsdT
A-96204.1
UGGAUcAAcAUUUUGGUUGdTsdT
AD-45887.1
A-96143.1
cuAcAuAuAAAcuAcAAGudTsdT
A-96144.1
ACUUGuAGUUuAuAUGuAGdTsdT
AD-45895.1
A-96177.1
GGGAGGcuuGAuGGAGAAudTsdT
A-96178.1
AUUCUCcAUcAAGCCUCCCdTsdT
AD-45902.1
A-96195.1
GGuGuuuucuAcuuGGGAudTsdT
A-96196.1
AUCCcAAGuAGAAAAcACCdTsdT
AD-45910.1
A-96229.1
AAGAGcAccAAGAAcuAcudTsdT
A-96230.1
AGuAGUUCUUGGUGCUCUUdTsdT
AD-45935.1
A-96239.1
uGGAGAAAAcAAccuAAAudTsdT
A-96240.1
AUUuAGGUUGUUUUCUCcAdTsdT
AD-45864.1
A-96151.1
GcAAcuAAcuAAcuuAAuudTsdT
A-96152.1
AAUuAAGUuAGUuAGUUGCdTsdT
AD-45873.1
A-96201.1
cAAccuAAAuGGuAAAuAudTsdT
A-96202.1
AuAUUuACcAUUuAGGUUGdTsdT
AD-45880.1
A-96125.1
GcuAuGuuAGAcGAuGuAAdTsdT
A-96126.1
UuAcAUCGUCuAAcAuAGCdTsdT
AD-45888.1
A-96159.1
cccAcAGAAAuuucucuAudTsdT
A-96160.1
AuAGAGAAAUUUCUGUGGGdTsdT
AD-45896.1
A-96193.1
GAuuuGGuGuuuucuAcuudTsdT
A-96194.1
AAGuAGAAAAcACcAAAUCdTsdT
AD-45903.1
A-96211.1
cAGAGccAAAAucAAGAuudTsdT
A-96212.1
AAUCUUGAUUUUGGCUCUGdTsdT
AD-45919.1
A-96217.1
AAAucAAGAuuuGcuAuGudTsdT
A-96218.1
AcAuAGcAAAUCUUGAUUUdTsdT
AD-45865.1
A-96167.1
cAuGGAcAuuAAuucAAcAdTsdT
A-96168.1
UGUUGAAUuAAUGUCcAUGdTsdT
AD-45874.1
A-96123.1
GAuuuGcuAuGuuAGAcGAdTsdT
A-96124.1
UCGUCuAAcAuAGcAAAUCdTsdT
AD-45881.1
A-96141.1
GAAcuAcAuAuAAAcuAcAdTsdT
A-96142.1
UGuAGUUuAuAUGuAGUUCdTsdT
AD-45889.1
A-96175.1
cGAAuAGAuGGAucAcAAAdTsdT
A-96176.1
UUUGUGAUCcAUCuAUUCGdTsdT
AD-45897.1
A-96209.1
cuuGuuAAAAcucuAAAcudTsdT
A-96210.1
AGUUuAGAGUUUuAAcAAGdTsdT
AD-45904.1
A-96227.1
AuuuGAucAGucuuuuuAudTsdT
A-96228.1
AuAAAAAGACUGAUcAAAUdTsdT
AD-45920.1
A-96233.1
uccAuGGAcAuuAAuucAAdTsdT
A-96234.1
UUGAAUuAAUGUCcAUGGAdTsdT
AD-45856.1
A-96117.1
cAcAAuuAAGcuccuucuudTsdT
A-96118.1
AAGAAGGAGCUuAAUUGUGdTsdT
AD-45929.1
A-96221.1
cAAcAuAuuuGAucAGucudTsdT
A-96222.1
AGACUGAUcAAAuAUGUUGdTsdT
AD-45866.1
A-96183.1
cuccAuAGuGAAGcAAucudTsdT
A-96184.1
AGAUUGCUUcACuAUGGAGdTsdT
AD-45875.1
A-96139.1
GccAAAuuAAuGAcAuAuudTsdT
A-96140.1
AAuAUGUcAUuAAUUUGGCdTsdT
AD-45882.1
A-96157.1
cAAcAGcAuAGucAAAuAAdTsdT
A-96158.1
UuAUUUGACuAUGCUGUUGdTsdT
AD-45890.1
A-96191.1
GGAAAucAcGAAAccAAcudTsdT
A-96192.1
AGUUGGUUUCGUGAUUUCCdTsdT
AD-45898.1
A-96131.1
cAGuuGGGAcAuGGucuuAdTsdT
A-96132.1
uAAGACcAUGUCCcAACUGdTsdT
AD-45857.1
A-96133.1
GAcAuGGucuuAAAGAcuudTsdT
A-96134.1
AAGUCUUuAAGACcAUGUCdTsdT
AD-45930.1
A-96237.1
uGuGGAGAAAAcAAccuAAdTsdT
A-96238.1
UuAGGUUGUUUUCUCcAcAdTsdT
AD-45867.1
A-96199.1
GuGGAGAAAAcAAccuAAAdTsdT
A-96200.1
UUuAGGUUGUUUUCUCcACdTsdT
AD-45876.1
A-96155.1
ccAAcAGcAuAGucAAAuAdTsdT
A-96156.1
uAUUUGACuAUGCUGUUGGdTsdT
AD-45883.1
A-96173.1
cAAcAucGAAuAGAuGGAudTsdT
A-96174.1
AUCcAUCuAUUCGAUGUUGdTsdT
AD-45891.1
A-96207.1
GcAAAuuuAAAAGGcAAuAdTsdT
A-96208.1
uAUUGCCUUUuAAAUUUGCdTsdT
AD-45914.1
A-96215.1
cAAAAucAAGAuuuGcuAudTsdT
A-96216.1
AuAGcAAAUCUUGAUUUUGdTsdT
AD-15838.1
A-26242.1
AGAGccAAAAucAAGAuuudTsdT
A-26243.2
AAAUCUuGAUUUuGGCUCUdTsdT
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; s is a phosphothiorate linkage.
TABLE 4
Results of single dose screen using ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide
duplexes listed in Table 3. The sequence of AD-15838.2 is
identical to the sequence of AD-15838.1. Delivery of siRNA
duplexes was done using LNPs. Human Hep3B
Duplex
10 nM
0.1 nM
STDEV, 10 nM
STDEV,0.1 nM
AD-15838.2
0.09
0.66
0.008
0.030
AD-45856.1
0.32
0.91
0.026
0.032
AD-45857.1
2.46
1.07
0.140
0.044
AD-45858.1
0.10
0.74
0.010
0.070
AD-45860.1
0.02
0.47
0.002
0.097
AD-45861.1
0.03
0.68
0.004
0.062
AD-45863.1
1.42
0.95
0.145
0.126
AD-45864.1
0.02
0.17
0.002
0.045
AD-45865.1
0.32
0.93
0.022
0.062
AD-45866.1
0.10
0.92
0.010
0.041
AD-45867.1
0.04
0.61
0.000
0.048
AD-45869.1
0.45
1.08
0.028
0.081
AD-45870.1
0.01
0.10
0.003
0.010
AD-45871.1
0.05
0.57
0.006
0.071
AD-45872.1
0.07
0.71
0.007
0.034
AD-45873.1
0.02
0.23
0.001
0.011
AD-45874.1
0.08
0.75
0.013
0.049
AD-45875.1
0.13
0.82
0.017
0.040
AD-45876.1
0.03
0.54
0.000
0.013
AD-45877.1
0.06
0.47
0.002
0.025
AD-45878.1
0.02
0.44
0.002
0.031
AD-45879.1
0.03
0.35
0.003
0.023
AD-45880.1
0.49
1.00
0.039
0.088
AD-45881.1
0.20
0.90
0.019
0.095
AD-45882.1
0.20
0.95
0.012
0.086
AD-45883.1
0.16
0.98
0.011
0.058
AD-45884.1
0.09
0.94
0.003
0.044
AD-45885.1
0.22
0.91
0.020
0.145
AD-45886.1
0.04
0.40
0.008
0.080
AD-45887.1
0.03
0.35
0.002
0.057
AD-45888.1
0.05
0.80
0.006
0.042
AD-45889.1
0.31
0.91
0.013
0.052
AD-45890.1
0.06
0.90
0.001
0.047
AD-45891.1
0.06
0.82
0.007
0.034
AD-45892.1
1.01
1.09
0.033
0.211
AD-45893.1
0.04
0.58
0.002
0.046
AD-45894.1
0.04
0.59
0.003
0.024
AD-45895.1
0.84
1.00
0.047
0.047
AD-45896.1
0.84
0.98
0.032
0.095
AD-45897.1
0.36
0.61
0.032
0.053
AD-45898.1
0.98
1.09
0.021
0.117
AD-45899.1
0.04
0.59
0.005
0.095
AD-45900.1
0.06
0.80
0.005
0.091
AD-45901.1
0.33
0.94
0.025
0.096
AD-45902.1
0.24
1.03
0.010
0.079
AD-45903.1
0.74
1.02
0.003
0.092
AD-45904.1
0.39
0.87
0.010
0.010
AD-45909.1
0.04
0.73
0.008
0.013
AD-45910.1
1.08
1.01
0.037
0.089
AD-45914.1
0.52
0.99
0.018
0.071
AD-45915.1
0.06
0.48
0.004
0.046
AD-45919.1
0.67
0.98
0.048
0.064
AD-45920.1
0.61
1.00
0.031
0.038
AD-45924.1
0.09
0.67
0.005
0.012
AD-45925.1
0.13
0.90
0.008
0.100
AD-45929.1
0.02
0.42
0.001
0.083
AD-45930.1
0.05
0.63
0.005
0.052
AD-45934.1
0.04
0.41
0.001
0.062
AD-45935.1
0.08
0.76
0.006
0.058
AD-45939.1
0.23
0.82
0.030
0.028
AD-1955.1
0.93
0.93
0.068
0.073
AD-1955.1
0.94
1.01
0.028
0.113
AD-1955.1
1.00
1.02
0.032
0.065
AD-1955.1
1.15
1.06
0.053
0.019
TABLE 5
Dose response screen results for ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide
duplexes listed in Table 3. The sequence of AD-15838.2 is identical
to the sequence of AD-15838.1.
Hep3B IC50
24 hrs
120 hrs
IC50
IC50
IC50 I
IC50 II
weighted
IC50 I
IC50 II
weighted
Duplex
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
AD-15838.2
0.027
0.006
0.017
0.657
0.937
0.800
AD-45860.1
0.006
0.002
0.004
0.045
0.032
0.039
AD-45864.1
0.002
0.001
0.002
0.046
0.042
0.044
AD-45870.1
0.002
0.001
0.001
0.011
0.008
0.010
AD-45873.1
0.005
0.004
0.005
0.037
0.025
0.031
AD-45876.1
0.032
0.006
0.019
0.269
0.045
0.156
AD-45877.1
0.018
0.012
0.015
1.660
0.538
1.091
AD-45878.1
0.023
0.015
0.019
0.252
0.131
0.190
AD-45879.1
0.002
0.003
0.003
0.023
0.029
0.026
AD-45886.1
0.004
0.004
0.004
0.030
0.018
0.025
AD-45887.1
0.010
0.009
0.010
0.058
0.059
0.059
AD-45915.1
0.016
0.015
0.015
0.110
0.056
0.083
AD-45929.1
0.023
0.008
0.016
0.227
0.025
0.124
AD-45934.1
0.006
0.006
0.006
0.110
0.045
0.077
TABLE 6
Results of cell viability screens using modified ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide duplexes listed in Table 3.
The sequence of AD-15838.2 is identical to the sequence of AD-15838.1. Viability data is
expressed as % viable relative to mock treated cells.
Ave
Ave
Ave
Ave
Ave
SD
SD
SD
SD
SD
Target
Duplex
10 nM
1 nM
500 pM
100 pM
50 pM
10 nM
1 nM
500 pM
100 pM
50 pM
HeLa day 3
ANGPTL3
AD-15838.2
37.34
58.67
70.92
89.86
94.98
9.45
12.28
15.06
22.37
18.23
ANGPTL3
AD-15838.2
29.13
48.99
63.18
79.21
94.47
1.62
5.56
4.34
11.15
11.31
ANGPTL3
AD-45860.1
67.10
75.49
77.93
86.57
90.51
6.99
12.93
6.39
6.97
3.57
ANGPTL3
AD-45864.1
99.13
96.95
86.77
89.20
84.36
7.90
7.22
12.60
4.85
6.87
ANGPTL3
AD-45870.1
82.36
97.02
95.33
95.67
92.27
8.07
5.12
7.97
7.05
10.29
ANGPTL3
AD-45873.1
67.96
90.01
90.60
94.20
103.63
11.26
22.61
15.92
22.92
16.97
ANGPTL3
AD-45876.1
64.00
76.71
80.21
81.71
91.23
6.60
13.94
10.15
10.81
13.89
ANGPTL3
AD-45877.1
79.55
77.33
79.98
91.96
93.46
1.66
9.80
8.73
16.63
11.41
ANGPTL3
AD-45878.1
81.95
78.22
78.74
87.93
85.03
15.37
22.72
22.59
30.84
40.04
ANGPTL3
AD-45878.1
66.83
70.71
82.14
82.80
83.14
17.48
6.49
6.86
19.92
21.15
ANGPTL3
AD-45879.1
37.56
45.55
59.28
76.35
78.38
3.50
7.96
19.73
34.33
33.99
ANGPTL3
AD-45886.1
72.75
57.90
64.51
81.92
82.89
14.73
12.64
11.78
25.60
23.14
ANGPTL3
AD-45887.1
38.01
53.91
59.31
76.44
85.73
0.58
10.81
6.27
11.12
10.92
ANGPTL3
AD-45915.1
48.06
52.17
67.90
95.45
100.77
8.13
15.15
29.11
32.49
38.79
ANGPTL3
AD-45929.1
29.27
44.58
52.87
76.45
88.03
4.17
9.67
14.49
31.74
28.82
ANGPTL3
AD-45934.1
68.20
64.11
76.92
79.57
92.11
15.79
11.25
19.99
26.08
26.30
(+) control
AD-19200
41.09
85.94
95.13
101.29
96.60
9.99
25.31
24.56
32.26
26.35
(+) control
AD-19200
23.99
72.76
86.51
108.10
111.13
5.35
34.52
29.24
35.99
31.88
(−) control
AD-1955
89.65
99.87
94.59
104.04
105.10
4.57
5.94
4.19
5.78
7.46
(−) control
AD-1955
104.74
99.78
105.79
109.19
108.08
10.94
7.74
11.12
7.91
10.30
(−) control
mock
100.00
6.92
(−) control
mock
100.00
9.85
(+) control
PLK
10.66
26.65
46.16
92.42
98.78
1.70
8.65
13.47
22.99
23.48
(+) control
PLK
10.74
11.41
17.33
61.02
86.59
3.39
2.61
1.49
27.42
37.31
HeLa day 6
ANGPTL3
AD-15838.2
47.94
80.97
90.44
94.37
96.10
29.05
25.12
13.62
8.88
4.72
ANGPTL3
AD-15838.2
40.32
83.80
89.88
95.94
98.27
22.47
16.51
10.03
3.83
4.19
ANGPTL3
AD-45860.1
57.38
84.84
88.90
96.74
94.03
24.55
17.35
9.67
3.17
6.58
ANGPTL3
AD-45864.1
98.65
100.87
101.13
96.86
98.24
4.35
1.91
2.22
3.41
1.80
ANGPTL3
AD-45870.1
92.69
98.71
98.49
100.07
99.28
3.94
2.67
2.36
1.19
2.65
ANGPTL3
AD-45873.1
91.78
97.38
98.81
97.57
96.22
12.47
6.26
4.08
6.22
8.64
ANGPTL3
AD-45876.1
63.54
85.68
92.13
96.48
95.97
14.74
16.50
10.03
5.81
7.51
ANGPTL3
AD-45877.1
94.17
93.21
96.39
96.70
96.98
7.12
8.00
4.58
3.05
6.15
ANGPTL3
AD-45878.1
66.46
85.75
89.73
94.60
96.59
8.20
7.41
5.27
3.21
3.91
ANGPTL3
AD-45878.1
70.80
89.30
92.54
96.60
95.09
5.18
2.13
1.61
0.50
4.15
ANGPTL3
AD-45879.1
8.29
48.25
73.54
87.47
92.19
4.66
20.05
16.04
9.06
7.90
ANGPTL3
AD-45886.1
23.69
60.65
78.49
93.41
94.15
8.19
13.90
7.15
3.35
4.06
ANGPTL3
AD-45887.1
7.24
26.03
57.68
95.99
98.80
3.07
13.10
14.94
1.40
2.54
ANGPTL3
AD-45915.1
10.38
58.38
85.69
97.24
99.76
6.83
15.66
8.39
1.33
4.15
ANGPTL3
AD-45929.1
11.73
36.67
51.90
76.71
85.08
4.80
14.19
15.34
12.37
10.60
ANGPTL3
AD-45934.1
73.57
88.48
92.94
91.50
95.97
5.36
2.96
5.50
5.44
4.39
(+) control
AD-19200
63.58
90.14
95.44
94.65
93.28
34.11
14.32
8.78
10.90
12.13
(+) control
AD-19200
16.05
78.65
85.78
93.09
96.22
9.77
15.57
19.50
13.34
10.96
(−) control
AD-1955
93.52
97.36
97.90
99.65
100.07
5.02
1.78
0.84
0.58
1.14
(−) control
AD-1955
75.39
93.61
97.79
99.60
100.96
8.37
2.50
2.27
2.68
3.16
(−) control
mock
100.00
1.32
(−) control
mock
100.00
3.35
(+) control
PLK
3.68
55.22
63.00
89.39
95.33
1.42
30.96
33.97
15.85
8.54
(+) control
PLK
2.69
3.74
9.74
67.07
82.96
0.15
0.96
3.60
22.70
19.34
Hep3B day 3
ANGPTL3
AD-15838.2
35.33
61.00
68.79
82.74
90.41
2.41
6.21
4.21
2.61
7.07
ANGPTL3
AD-15838.2
35.34
61.04
72.14
89.71
106.88
1.49
2.61
7.37
6.48
7.13
ANGPTL3
AD-45860.1
17.79
39.25
60.57
94.28
99.85
1.07
3.51
3.57
13.09
16.41
ANGPTL3
AD-45864.1
80.35
88.19
87.01
89.39
92.09
6.93
6.98
9.42
7.41
17.05
ANGPTL3
AD-45870.1
75.00
93.30
96.64
106.29
99.08
7.10
12.24
4.01
5.95
9.64
ANGPTL3
AD-45873.1
42.68
78.45
82.26
97.11
96.58
5.17
5.04
8.31
12.11
11.33
ANGPTL3
AD-45876.1
31.37
55.00
70.69
93.49
91.00
4.39
6.09
5.47
15.11
6.38
ANGPTL3
AD-45877.1
74.45
94.60
96.70
103.77
106.75
3.27
2.44
3.45
6.10
7.40
ANGPTL3
AD-45878.1
50.22
69.65
80.49
92.77
97.37
2.51
14.94
10.44
8.21
5.30
ANGPTL3
AD-45878.1
44.85
65.39
75.67
92.83
109.67
10.10
7.76
8.56
7.78
4.97
ANGPTL3
AD-45879.1
23.73
60.81
84.59
95.72
108.68
6.43
21.36
19.62
13.69
5.95
ANGPTL3
AD-45886.1
27.19
55.35
64.97
100.18
102.09
0.97
6.65
11.46
6.91
4.08
ANGPTL3
AD-45887.1
41.70
97.18
101.91
111.27
105.18
9.26
6.81
7.36
1.72
2.23
ANGPTL3
AD-45915.1
45.10
66.31
82.22
97.97
103.30
6.91
11.84
14.79
6.54
2.48
ANGPTL3
AD-45929.1
48.58
79.14
89.96
95.00
101.37
10.40
10.29
10.52
18.24
10.53
ANGPTL3
AD-45934.1
80.15
102.93
112.82
114.16
113.98
5.28
0.62
4.19
0.75
3.99
(+) control
AD-19200
14.79
55.23
72.90
89.64
94.30
2.17
5.42
7.19
10.28
16.39
(+) control
AD-19200
22.76
92.02
101.56
106.68
113.09
6.61
18.99
7.41
9.83
10.64
(−) control
AD-1955
77.77
81.25
82.23
88.21
95.02
2.83
5.40
5.08
5.42
6.63
(−) control
AD-1955
80.42
86.70
90.23
93.46
97.04
10.53
5.70
8.14
3.27
3.45
(−) control
mock
100.00
5.77
(−) control
mock
100.00
9.79
(+) control
PLK
10.91
12.89
14.31
23.87
50.93
0.17
0.87
1.64
1.13
7.80
(+) control
PLK
13.19
16.12
22.89
55.03
94.35
0.78
0.88
8.36
18.88
9.85
Hep3B day 6
ANGPTL3
AD-15838.2
78.88
89.58
93.08
91.10
100.66
11.60
9.15
12.04
10.51
5.87
ANGPTL3
AD-15838.2
81.17
85.91
87.27
103.95
103.59
7.75
3.29
8.07
7.93
9.82
ANGPTL3
AD-45860.1
84.11
87.77
93.22
99.15
96.75
14.22
13.36
20.98
13.15
17.62
ANGPTL3
AD-45864.1
99.27
111.82
106.28
99.15
97.55
7.77
16.31
14.24
15.40
9.18
ANGPTL3
AD-45870.1
95.49
109.60
104.16
104.65
106.76
11.92
12.98
9.25
10.29
19.12
ANGPTL3
AD-45873.1
71.45
90.62
93.44
102.07
107.72
4.71
4.40
15.02
11.96
10.16
ANGPTL3
AD-45876.1
76.92
82.09
89.44
95.27
105.41
9.39
13.55
7.93
9.77
10.42
ANGPTL3
AD-45877.1
82.98
98.05
95.07
103.55
104.14
11.22
13.45
1.27
8.88
6.49
ANGPTL3
AD-45878.1
75.14
82.48
89.68
92.71
95.72
8.65
10.07
10.77
12.44
15.04
ANGPTL3
AD-45878.1
65.90
77.37
78.33
84.54
99.49
10.21
13.22
9.95
11.65
11.17
ANGPTL3
AD-45879.1
86.42
89.45
101.50
97.30
100.66
10.59
10.12
19.77
13.19
9.54
ANGPTL3
AD-45886.1
91.15
79.31
80.76
86.52
94.04
12.89
11.88
5.38
4.92
6.80
ANGPTL3
AD-45887.1
91.67
103.38
107.88
100.05
102.05
10.80
14.84
19.18
13.72
18.00
ANGPTL3
AD-45915.1
81.97
85.91
91.81
94.95
102.13
18.49
19.30
7.19
12.72
16.64
ANGPTL3
AD-45929.1
61.92
79.39
87.28
88.09
96.00
6.80
10.76
5.80
10.68
16.66
ANGPTL3
AD-45934.1
85.84
89.66
97.67
99.91
102.54
12.39
14.25
4.74
9.51
4.28
(+) control
AD-19200
50.48
65.62
79.67
98.61
96.87
4.60
4.64
7.20
5.08
7.37
(+) control
AD-19200
52.01
75.89
92.59
101.47
99.66
4.35
20.87
13.57
6.50
11.76
(−) control
AD-1955
91.77
95.87
93.06
95.10
97.52
8.87
3.46
1.46
2.00
3.84
(−) control
AD-1955
93.65
94.41
89.42
100.59
103.91
9.91
14.90
6.80
11.99
10.31
(−) control
mock
100.00
5.10
(−) control
mock
100.00
7.35
(+) control
PLK
36.43
37.75
40.19
55.25
64.59
3.44
2.75
3.65
5.33
5.02
(+) control
PLK
38.70
43.68
50.32
75.17
89.62
3.40
3.85
8.10
10.54
10.69
TABLE 7
Unmodified sense and antisense strand sequences
of ANGPTL3 GalNac-conjugated dsRNAs
Sense Sequence
(SEQ ID NOS 264-448,
Sense
respectively, in order
Position in
Duplex ID
Name
of appearance)
NM_014495.2
AD-53063.1
A-108558.1
AAAGACAACAAACAUUAUAUUx
1066-1086
AD-52965.1
A-108310.1
ACAAUUAAGCUCCUUCUUUUUx
58-78
AD-53030.1
A-108410.1
UGUCACUUGAACUCAACUCAAx
398-418
AD-52953.1
A-108306.1
UCACAAUUAAGCUCCUUCUUUx
56-76
AD-53001.1
A-108416.1
CUUGAACUCAACUCAAAACUUx
403-423
AD-53080.1
A-108548.1
CUCCAUAGUGAAGCAAUCUAAx
1014-1034
AD-52971.1
A-108312.1
CAAUUAAGCUCCUUCUUUUUAx
59-79
AD-53071.1
A-108498.1
ACCCAGCAACUCUCAAGUUUUx
840-860
AD-53024.1
A-108408.1
GAAUAUGUCACUUGAACUCAAx
393-413
AD-52977.1
A-108314.1
AAUUAAGCUCCUUCUUUUUAUx
60-80
AD-53064.1
A-108574.1
CAUUAUAUUGAAUAUUCUUUUx
1078-1098
AD-53033.1
A-108458.1
ACUAACUAACUUAAUUCAAAAx
483-503
AD-52954.1
A-108322.1
UUAUUGUUCCUCUAGUUAUUUx
77-97
AD-53098.1
A-108554.1
CAUAGUGAAGCAAUCUAAUUAx
1017-1037
AD-53092.1
A-108552.1
CCAUAGUGAAGCAAUCUAAUUx
1016-1036
AD-53073.1
A-108530.1
GAUCACAAAACUUCAAUGAAAx
923-943
AD-53132.1
A-108628.1
AUGGAAGGUUAUACUCUAUAAx
1364-1384
AD-53086.1
A-108550.1
UCCAUAGUGAAGCAAUCUAAUx
1015-1035
AD-52961.1
A-108340.1
CUAUGUUAGACGAUGUAAAAAx
164-184
AD-52983.1
A-108316.1
AUUAAGCUCCUUCUUUUUAUUx
61-81
AD-53027.1
A-108456.1
AACUAACUAACUUAAUUCAAAx
482-502
AD-52986.1
A-108364.1
GGCCAAAUUAAUGACAUAUUUx
247-267
AD-52989.1
A-108318.1
UUUUAUUGUUCCUCUAGUUAUx
75-95
AD-52981.1
A-108378.1
ACAUAUUUGAUCAGUCUUUUUx
278-298
AD-53077.1
A-108500.1
CCCAGCAACUCUCAAGUUUUUx
841-861
AD-53095.1
A-108506.1
CAGGUAGUCCAUGGACAUUAAx
884-904
AD-52970.1
A-108390.1
ACUGAGAAGAACUACAUAUAAx
345-365
AD-53015.1
A-108452.1
GAGCAACUAACUAACUUAAUUx
478-498
AD-53147.1
A-108618.1
AACAACCUAAAUGGUAAAUAUx
1282-1302
AD-53103.1
A-108540.1
CCUAGAGAAGAUAUACUCCAUx
999-1019
AD-52969.1
A-108374.1
CAACAUAUUUGAUCAGUCUUUx
276-296
AD-53075.1
A-108562.1
ACAACAAACAUUAUAUUGAAUx
1070-1090
AD-52994.1
A-108398.1
ACAUAUAAACUACAAGUCAAAx
358-378
AD-52960.1
A-108324.1
CUAGUUAUUUCCUCCAGAAUUx
88-108
AD-53003.1
A-108448.1
AAGAGCAACUAACUAACUUAAx
476-496
AD-52995.1
A-108320.1
UUUAUUGUUCCUCUAGUUAUUx
76-96
AD-53037.1
A-108428.1
CUCCUAGAAGAAAAAAUUCUAx
430-450
AD-53087.1
A-108566.1
AACAAACAUUAUAUUGAAUAUx
1072-1092
AD-53076.1
A-108578.1
GGAAAUCACGAAACCAACUAUx
1105-1125
AD-52975.1
A-108376.1
AACAUAUUUGAUCAGUCUUUUx
277-297
AD-53138.1
A-108630.1
UGGAAGGUUAUACUCUAUAAAx
1365-1385
AD-53091.1
A-108536.1
GGAGAACUACAAAUAUGGUUUx
948-968
AD-53124.1
A-108594.1
GAAAACAAAGAUUUGGUGUUUx
1174-1194
AD-53125.1
A-108610.1
AGUGUGGAGAAAACAACCUAAx
1271-1291
AD-53036.1
A-108412.1
GUCACUUGAACUCAACUCAAAx
399-419
AD-53061.1
A-108526.1
GAUGGAUCACAAAACUUCAAUx
919-939
AD-53093.1
A-108568.1
ACAAACAUUAUAUUGAAUAUUx
1073-1093
AD-53137.1
A-108614.1
UGUGGAGAAAACAACCUAAAUx
1273-1293
AD-52999.1
A-108384.1
AUCAGUCUUUUUAUGAUCUAUx
287-307
AD-53069.1
A-108560.1
GACAACAAACAUUAUAUUGAAx
1069-1089
AD-53034.1
A-108474.1
CAACAGCAUAGUCAAAUAAAAx
622-642
AD-52976.1
A-108392.1
CUGAGAAGAACUACAUAUAAAx
346-366
AD-52996.1
A-108336.1
UGCUAUGUUAGACGAUGUAAAx
162-182
AD-53029.1
A-108488.1
AACCCACAGAAAUUUCUCUAUx
680-700
AD-53020.1
A-108438.1
CUUCAACAAAAAGUGAAAUAUx
451-471
AD-53042.1
A-108414.1
UCACUUGAACUCAACUCAAAAx
400-420
AD-53011.1
A-108482.1
CAUAGUCAAAUAAAAGAAAUAx
628-648
AD-52957.1
A-108370.1
CAAAAACUCAACAUAUUUGAUx
268-288
AD-53008.1
A-108434.1
UACUUCAACAAAAAGUGAAAUx
449-469
AD-53065.1
A-108496.1
GACCCAGCAACUCUCAAGUUUx
839-859
AD-53115.1
A-108638.1
UUGAAUGAACUGAGGCAAAUUx
1427-1447
AD-53012.1
A-108404.1
UAUAAACUACAAGUCAAAAAUx
361-381
AD-53004.1
A-108464.1
AAACAAGAUAAUAGCAUCAAAx
559-579
AD-53021.1
A-108454.1
CAACUAACUAACUUAAUUCAAx
481-501
AD-52955.1
A-108338.1
GCUAUGUUAGACGAUGUAAAAx
163-183
AD-53119.1
A-108608.1
ACUUGGGAUCACAAAGCAAAAx
1198-1218
AD-52990.1
A-108334.1
UUGCUAUGUUAGACGAUGUAAx
161-181
AD-52964.1
A-108388.1
AACUGAGAAGAACUACAUAUAx
344-364
AD-52973.1
A-108344.1
GAUGUAAAAAUUUUAGCCAAUx
175-195
AD-53074.1
A-108546.1
ACUCCAUAGUGAAGCAAUCUAx
1013-1033
AD-53026.1
A-108440.1
UUCAACAAAAAGUGAAAUAUUx
452-472
AD-53062.1
A-108542.1
CUAGAGAAGAUAUACUCCAUAx
1000-1020
AD-53114.1
A-108622.1
CAACCUAAAUGGUAAAUAUAAx
1284-1304
AD-53082.1
A-108580.1
GAAAUCACGAAACCAACUAUAx
1106-1126
AD-53035.1
A-108490.1
CCACAGAAAUUUCUCUAUCUUx
683-703
AD-52978.1
A-108330.1
AAAUCAAGAUUUGCUAUGUUAx
151-171
AD-53084.1
A-108518.1
ACAUUAAUUCAACAUCGAAUAx
898-918
AD-52972.1
A-108328.1
CCAGAGCCAAAAUCAAGAUUUx
142-162
AD-53002.1
A-108432.1
CUACUUCAACAAAAAGUGAAAx
448-468
AD-53078.1
A-108516.1
GACAUUAAUUCAACAUCGAAUx
897-917
AD-53072.1
A-108514.1
GGACAUUAAUUCAACAUCGAAx
896-916
AD-53005.1
A-108480.1
GCAUAGUCAAAUAAAAGAAAUx
627-647
AD-53083.1
A-108502.1
CUCUCAAGUUUUUCAUGUCUAx
849-869
AD-53102.1
A-108524.1
AUCGAAUAGAUGGAUCACAAAx
911-931
AD-53105.1
A-108572.1
ACAUUAUAUUGAAUAUUCUUUx
1077-1097
AD-53090.1
A-108520.1
UUAAUUCAACAUCGAAUAGAUx
901-921
AD-53010.1
A-108466.1
GAUAAUAGCAUCAAAGACCUUx
565-585
AD-52998.1
A-108368.1
UGACAUAUUUCAAAAACUCAAx
258-278
AD-52992.1
A-108366.1
AAAUUAAUGACAUAUUUCAAAx
251-271
AD-53068.1
A-108544.1
GAAGAUAUACUCCAUAGUGAAx
1005-1025
AD-53032.1
A-108442.1
AAUAUUUAGAAGAGCAACUAAx
467-487
AD-52967.1
A-108342.1
CGAUGUAAAAAUUUUAGCCAAx
174-194
AD-53096.1
A-108522.1
UUCAACAUCGAAUAGAUGGAUx
905-925
AD-53131.1
A-108612.1
GUGUGGAGAAAACAACCUAAAx
1272-1292
AD-52963.1
A-108372.1
UCAACAUAUUUGAUCAGUCUUx
275-295
AD-53089.1
A-108504.1
UCAGGUAGUCCAUGGACAUUAx
883-903
AD-53044.1
A-108446.1
UUUAGAAGAGCAACUAACUAAx
471-491
AD-52988.1
A-108396.1
UACAUAUAAACUACAAGUCAAx
357-377
AD-53067.1
A-108528.1
GGAUCACAAAACUUCAAUGAAx
922-942
AD-53009.1
A-108450.1
AGAGCAACUAACUAACUUAAUx
477-497
AD-53022.1
A-108470.1
ACCAACAGCAUAGUCAAAUAAx
620-640
AD-53016.1
A-108468.1
AACCAACAGCAUAGUCAAAUAx
619-639
AD-53007.1
A-108418.1
GAACUCAACUCAAAACUUGAAx
406-426
AD-53148.1
A-108634.1
UACUCUAUAAAAUCAACCAAAx
1375-1395
AD-53040.1
A-108476.1
CAGCAUAGUCAAAUAAAAGAAx
625-645
AD-53041.1
A-108492.1
GAAAUAAGAAAUGUAAAACAUx
748-768
AD-53039.1
A-108460.1
CUAACUAACUUAAUUCAAAAUx
484-504
AD-53139.1
A-108646.1
AUGAACUGAGGCAAAUUUAAAx
1431-1451
AD-53144.1
A-108648.1
UGAACUGAGGCAAAUUUAAAAx
1432-1452
AD-53142.1
A-108616.1
AAACAACCUAAAUGGUAAAUAx
1281-1301
AD-53108.1
A-108620.1
ACAACCUAAAUGGUAAAUAUAx
1283-1303
AD-53079.1
A-108532.1
AACGUGGGAGAACUACAAAUAx
942-962
AD-53133.1
A-108644.1
AAUGAACUGAGGCAAAUUUAAx
1430-1450
AD-53104.1
A-108556.1
GUUGGAAGACUGGAAAGACAAx
1053-1073
AD-53088.1
A-108582.1
UGGCAAUGUCCCCAAUGCAAUx
1149-1169
AD-53101.1
A-108508.1
GGUAGUCCAUGGACAUUAAUUx
886-906
AD-53000.1
A-108400.1
CAUAUAAACUACAAGUCAAAAx
359-379
AD-53112.1
A-108590.1
AAUCCCGGAAAACAAAGAUUUx
1167-1187
AD-53107.1
A-108604.1
CUACUUGGGAUCACAAAGCAAx
1196-1216
AD-53121.1
A-108640.1
UGAAUGAACUGAGGCAAAUUUx
1428-1448
AD-53046.1
A-108478.1
AGCAUAGUCAAAUAAAAGAAAx
626-646
AD-53038.1
A-108444.1
AUUUAGAAGAGCAACUAACUAx
470-490
AD-53140.1
A-108662.1
AGGCAAAUUUAAAAGGCAAUAx
1439-1459
AD-52987.1
A-108380.1
CAUAUUUGAUCAGUCUUUUUAx
279-299
AD-53130.1
A-108596.1
AAAACAAAGAUUUGGUGUUUUx
1175-1195
AD-53106.1
A-108588.1
CAAUCCCGGAAAACAAAGAUUx
1166-1186
AD-53081.1
A-108564.1
CAACAAACAUUAUAUUGAAUAx
1071-1091
AD-53118.1
A-108592.1
GGAAAACAAAGAUUUGGUGUUx
1173-1193
AD-53136.1
A-108598.1
ACAAAGAUUUGGUGUUUUCUAx
1178-1198
AD-53127.1
A-108642.1
GAAUGAACUGAGGCAAAUUUAx
1429-1449
AD-53066.1
A-108512.1
CCAUGGACAUUAAUUCAACAUx
892-912
AD-53013.1
A-108420.1
AACUCAACUCAAAACUUGAAAx
407-427
AD-52991.1
A-108350.1
CAGUUGGGACAUGGUCUUAAAx
205-225
AD-53099.1
A-108570.1
AACAUUAUAUUGAAUAUUCUUx
1076-1096
AD-52958.1
A-108386.1
ACCAGUGAAAUCAAAGAAGAAx
316-336
AD-53097.1
A-108538.1
GUUGGGCCUAGAGAAGAUAUAx
993-1013
AD-52966.1
A-108326.1
CUCCAGAGCCAAAAUCAAGAUx
140-160
AD-53145.1
A-108664.1
GGCAAAUUUAAAAGGCAAUAAx
1440-1460
AD-53113.1
A-108606.1
UACUUGGGAUCACAAAGCAAAx
1197-1217
AD-52993.1
A-108382.1
GAUCAGUCUUUUUAUGAUCUAx
286-306
AD-53031.1
A-108426.1
GAAAGCCUCCUAGAAGAAAAAx
424-444
AD-53017.1
A-108484.1
AGUCAAAUAAAAGAAAUAGAAx
631-651
AD-53143.1
A-108632.1
AUACUCUAUAAAAUCAACCAAx
1374-1394
AD-53149.1
A-108650.1
GAACUGAGGCAAAUUUAAAAAx
1433-1453
AD-53059.1
A-108494.1
AGACCCAGCAACUCUCAAGUUx
838-858
AD-53006.1
A-108402.1
AUAUAAACUACAAGUCAAAAAx
360-380
AD-53025.1
A-108424.1
UGAAAGCCUCCUAGAAGAAAAx
423-443
AD-53085.1
A-108534.1
GGGAGAACUACAAAUAUGGUUx
947-967
AD-52984.1
A-108332.1
AGAUUUGCUAUGUUAGACGAUx
157-177
AD-53023.1
A-108486.1
GAACCCACAGAAAUUUCUCUAx
679-699
AD-53014.1
A-108436.1
ACUUCAACAAAAAGUGAAAUAx
450-470
AD-53060.1
A-108510.1
AGUCCAUGGACAUUAAUUCAAx
889-909
AD-53110.1
A-108652.1
AACUGAGGCAAAUUUAAAAGAx
1434-1454
AD-52980.1
A-108362.1
GGGCCAAAUUAAUGACAUAUUx
246-266
AD-53109.1
A-108636.1
AUCCAUCCAACAGAUUCAGAAx
1402-1422
AD-53141.1
A-108600.1
AAGAUUUGGUGUUUUCUACUUx
1181-1201
AD-53126.1
A-108626.1
GUCUCAAAAUGGAAGGUUAUAx
1356-1376
AD-53116.1
A-108654.1
ACUGAGGCAAAUUUAAAAGGAx
1435-1455
AD-52997.1
A-108352.1
GGGACAUGGUCUUAAAGACUUx
210-230
AD-53120.1
A-108624.1
AUGGUAAAUAUAACAAACCAAx
1292-1312
AD-53070.1
A-108576.1
GGGAAAUCACGAAACCAACUAx
1104-1124
AD-53028.1
A-108472.1
CCAACAGCAUAGUCAAAUAAAx
621-641
AD-53146.1
A-108602.1
UUUUCUACUUGGGAUCACAAAx
1192-1212
AD-52982.1
A-108394.1
AGAACUACAUAUAAACUACAAx
352-372
AD-53111.1
A-108668.1
AGAGUAUGUGUAAAAAUCUGUx
1915-1935
AD-53045.1
A-108462.1
AAAACAAGAUAAUAGCAUCAAx
558-578
AD-53123.1
A-108672.1
AGUAUGUGUAAAAAUCUGUAAx
1917-1937
AD-53018.1
A-108406.1
AGUCAAAAAUGAAGAGGUAAAx
372-392
AD-52956.1
A-108354.1
GGACAUGGUCUUAAAGACUUUx
211-231
AD-53134.1
A-108660.1
GAGGCAAAUUUAAAAGGCAAUx
1438-1458
AD-52968.1
A-108358.1
GUCUUAAAGACUUUGUCCAUAx
218-238
AD-53122.1
A-108656.1
CUGAGGCAAAUUUAAAAGGCAx
1436-1456
AD-53100.1
A-108586.1
GCAAUCCCGGAAAACAAAGAUx
1165-1185
AD-53128.1
A-108658.1
UGAGGCAAAUUUAAAAGGCAAx
1437-1457
AD-53043.1
A-108430.1
UCUACUUCAACAAAAAGUGAAx
447-467
AD-53135.1
A-108676.1
UAUGUGUAAAAAUCUGUAAUAx
1919-1939
AD-53094.1
A-108584.1
AAUGCAAUCCCGGAAAACAAAx
1162-1182
AD-53019.1
A-108422.1
CUUGAAAGCCUCCUAGAAGAAx
421-441
AD-53129.1
A-108674.1
GUAUGUGUAAAAAUCUGUAAUx
1918-1938
AD-53150.1
A-108666.1
CAGAGUAUGUGUAAAAAUCUUx
1914-1934
AD-53117.1
A-108670.1
GAGUAUGUGUAAAAAUCUGUAx
1916-1936
AD-52985.1
A-108348.1
UCAGUUGGGACAUGGUCUUAAx
204-224
AD-52962.1
A-108356.1
GGUCUUAAAGACUUUGUCCAUx
217-237
AD-52974.1
A-108360.1
UCUUAAAGACUUUGUCCAUAAx
219-239
AD-52979.1
A-108346.1
UUCAGUUGGGACAUGGUCUUAx
203-223
Antisense Sequence
(SEQ ID NOS 449-633,
Antisense
respectively,
Position in
Duplex ID
Name
in order of appearance)
NM_014495.2
AD-53063.1
A-108559.1
AAUAUAAUGUUUGUUGUCUUUCC
1064-1086
AD-52965.1
A-108311.1
AAAAAGAAGGAGCUUAAUUGUGA
56-78
AD-53030.1
A-108411.1
UUGAGUUGAGUUCAAGUGACAUA
396-418
AD-52953.1
A-108307.1
AAAGAAGGAGCUUAAUUGUGAAC
54-76
AD-53001.1
A-108417.1
AAGUUUUGAGUUGAGUUCAAGUG
401-423
AD-53080.1
A-108549.1
UUAGAUUGCUUCACUAUGGAGUA
1012-1034
AD-52971.1
A-108313.1
UAAAAAGAAGGAGCUUAAUUGUG
57-79
AD-53071.1
A-108499.1
AAAACUUGAGAGUUGCUGGGUCU
838-860
AD-53024.1
A-108409.1
UUGAGUUCAAGUGACAUAUUCUU
391-413
AD-52977.1
A-108315.1
AUAAAAAGAAGGAGCUUAAUUGU
58-80
AD-53064.1
A-108575.1
AAAAGAAUAUUCAAUAUAAUGUU
1076-1098
AD-53033.1
A-108459.1
UUUUGAAUUAAGUUAGUUAGUUG
481-503
AD-52954.1
A-108323.1
AAAUAACUAGAGGAACAAUAAAA
75-97
AD-53098.1
A-108555.1
UAAUUAGAUUGCUUCACUAUGGA
1015-1037
AD-53092.1
A-108553.1
AAUUAGAUUGCUUCACUAUGGAG
1014-1036
AD-53073.1
A-108531.1
UUUCAUUGAAGUUUUGUGAUCCA
921-943
AD-53132.1
A-108629.1
UUAUAGAGUAUAACCUUCCAUUU
1362-1384
AD-53086.1
A-108551.1
AUUAGAUUGCUUCACUAUGGAGU
1013-1035
AD-52961.1
A-108341.1
UUUUUACAUCGUCUAACAUAGCA
162-184
AD-52983.1
A-108317.1
AAUAAAAAGAAGGAGCUUAAUUG
59-81
AD-53027.1
A-108457.1
UUUGAAUUAAGUUAGUUAGUUGC
480-502
AD-52986.1
A-108365.1
AAAUAUGUCAUUAAUUUGGCCCU
245-267
AD-52989.1
A-108319.1
AUAACUAGAGGAACAAUAAAAAG
73-95
AD-52981.1
A-108379.1
AAAAAGACUGAUCAAAUAUGUUG
276-298
AD-53077.1
A-108501.1
AAAAACUUGAGAGUUGCUGGGUC
839-861
AD-53095.1
A-108507.1
UUAAUGUCCAUGGACUACCUGAU
882-904
AD-52970.1
A-108391.1
UUAUAUGUAGUUCUUCUCAGUUC
343-365
AD-53015.1
A-108453.1
AAUUAAGUUAGUUAGUUGCUCUU
476-498
AD-53147.1
A-108619.1
AUAUUUACCAUUUAGGUUGUUUU
1280-1302
AD-53103.1
A-108541.1
AUGGAGUAUAUCUUCUCUAGGCC
997-1019
AD-52969.1
A-108375.1
AAAGACUGAUCAAAUAUGUUGAG
274-296
AD-53075.1
A-108563.1
AUUCAAUAUAAUGUUUGUUGUCU
1068-1090
AD-52994.1
A-108399.1
UUUGACUUGUAGUUUAUAUGUAG
356-378
AD-52960.1
A-108325.1
AAUUCUGGAGGAAAUAACUAGAG
86-108
AD-53003.1
A-108449.1
UUAAGUUAGUUAGUUGCUCUUCU
474-496
AD-52995.1
A-108321.1
AAUAACUAGAGGAACAAUAAAAA
74-96
AD-53037.1
A-108429.1
UAGAAUUUUUUCUUCUAGGAGGC
428-450
AD-53087.1
A-108567.1
AUAUUCAAUAUAAUGUUUGUUGU
1070-1092
AD-53076.1
A-108579.1
AUAGUUGGUUUCGUGAUUUCCCA
1103-1125
AD-52975.1
A-108377.1
AAAAGACUGAUCAAAUAUGUUGA
275-297
AD-53138.1
A-108631.1
UUUAUAGAGUAUAACCUUCCAUU
1363-1385
AD-53091.1
A-108537.1
AAACCAUAUUUGUAGUUCUCCCA
946-968
AD-53124.1
A-108595.1
AAACACCAAAUCUUUGUUUUCCG
1172-1194
AD-53125.1
A-108611.1
UUAGGUUGUUUUCUCCACACUCA
1269-1291
AD-53036.1
A-108413.1
UUUGAGUUGAGUUCAAGUGACAU
397-419
AD-53061.1
A-108527.1
AUUGAAGUUUUGUGAUCCAUCUA
917-939
AD-53093.1
A-108569.1
AAUAUUCAAUAUAAUGUUUGUUG
1071-1093
AD-53137.1
A-108615.1
AUUUAGGUUGUUUUCUCCACACU
1271-1293
AD-52999.1
A-108385.1
AUAGAUCAUAAAAAGACUGAUCA
285-307
AD-53069.1
A-108561.1
UUCAAUAUAAUGUUUGUUGUCUU
1067-1089
AD-53034.1
A-108475.1
UUUUAUUUGACUAUGCUGUUGGU
620-642
AD-52976.1
A-108393.1
UUUAUAUGUAGUUCUUCUCAGUU
344-366
AD-52996.1
A-108337.1
UUUACAUCGUCUAACAUAGCAAA
160-182
AD-53029.1
A-108489.1
AUAGAGAAAUUUCUGUGGGUUCU
678-700
AD-53020.1
A-108439.1
AUAUUUCACUUUUUGUUGAAGUA
449-471
AD-53042.1
A-108415.1
UUUUGAGUUGAGUUCAAGUGACA
398-420
AD-53011.1
A-108483.1
UAUUUCUUUUAUUUGACUAUGCU
626-648
AD-52957.1
A-108371.1
AUCAAAUAUGUUGAGUUUUUGAA
266-288
AD-53008.1
A-108435.1
AUUUCACUUUUUGUUGAAGUAGA
447-469
AD-53065.1
A-108497.1
AAACUUGAGAGUUGCUGGGUCUG
837-859
AD-53115.1
A-108639.1
AAUUUGCCUCAGUUCAUUCAAAG
1425-1447
AD-53012.1
A-108405.1
AUUUUUGACUUGUAGUUUAUAUG
359-381
AD-53004.1
A-108465.1
UUUGAUGCUAUUAUCUUGUUUUU
557-579
AD-53021.1
A-108455.1
UUGAAUUAAGUUAGUUAGUUGCU
479-501
AD-52955.1
A-108339.1
UUUUACAUCGUCUAACAUAGCAA
161-183
AD-53119.1
A-108609.1
UUUUGCUUUGUGAUCCCAAGUAG
1196-1218
AD-52990.1
A-108335.1
UUACAUCGUCUAACAUAGCAAAU
159-181
AD-52964.1
A-108389.1
UAUAUGUAGUUCUUCUCAGUUCC
342-364
AD-52973.1
A-108345.1
AUUGGCUAAAAUUUUUACAUCGU
173-195
AD-53074.1
A-108547.1
UAGAUUGCUUCACUAUGGAGUAU
1011-1033
AD-53026.1
A-108441.1
AAUAUUUCACUUUUUGUUGAAGU
450-472
AD-53062.1
A-108543.1
UAUGGAGUAUAUCUUCUCUAGGC
998-1020
AD-53114.1
A-108623.1
UUAUAUUUACCAUUUAGGUUGUU
1282-1304
AD-53082.1
A-108581.1
UAUAGUUGGUUUCGUGAUUUCCC
1104-1126
AD-53035.1
A-108491.1
AAGAUAGAGAAAUUUCUGUGGGU
681-703
AD-52978.1
A-108331.1
UAACAUAGCAAAUCUUGAUUUUG
149-171
AD-53084.1
A-108519.1
UAUUCGAUGUUGAAUUAAUGUCC
896-918
AD-52972.1
A-108329.1
AAAUCUUGAUUUUGGCUCUGGAG
140-162
AD-53002.1
A-108433.1
UUUCACUUUUUGUUGAAGUAGAA
446-468
AD-53078.1
A-108517.1
AUUCGAUGUUGAAUUAAUGUCCA
895-917
AD-53072.1
A-108515.1
UUCGAUGUUGAAUUAAUGUCCAU
894-916
AD-53005.1
A-108481.1
AUUUCUUUUAUUUGACUAUGCUG
625-647
AD-53083.1
A-108503.1
UAGACAUGAAAAACUUGAGAGUU
847-869
AD-53102.1
A-108525.1
UUUGUGAUCCAUCUAUUCGAUGU
909-931
AD-53105.1
A-108573.1
AAAGAAUAUUCAAUAUAAUGUUU
1075-1097
AD-53090.1
A-108521.1
AUCUAUUCGAUGUUGAAUUAAUG
899-921
AD-53010.1
A-108467.1
AAGGUCUUUGAUGCUAUUAUCUU
563-585
AD-52998.1
A-108369.1
UUGAGUUUUUGAAAUAUGUCAUU
256-278
AD-52992.1
A-108367.1
UUUGAAAUAUGUCAUUAAUUUGG
249-271
AD-53068.1
A-108545.1
UUCACUAUGGAGUAUAUCUUCUC
1003-1025
AD-53032.1
A-108443.1
UUAGUUGCUCUUCUAAAUAUUUC
465-487
AD-52967.1
A-108343.1
UUGGCUAAAAUUUUUACAUCGUC
172-194
AD-53096.1
A-108523.1
AUCCAUCUAUUCGAUGUUGAAUU
903-925
AD-53131.1
A-108613.1
UUUAGGUUGUUUUCUCCACACUC
1270-1292
AD-52963.1
A-108373.1
AAGACUGAUCAAAUAUGUUGAGU
273-295
AD-53089.1
A-108505.1
UAAUGUCCAUGGACUACCUGAUA
881-903
AD-53044.1
A-108447.1
UUAGUUAGUUGCUCUUCUAAAUA
469-491
AD-52988.1
A-108397.1
UUGACUUGUAGUUUAUAUGUAGU
355-377
AD-53067.1
A-108529.1
UUCAUUGAAGUUUUGUGAUCCAU
920-942
AD-53009.1
A-108451.1
AUUAAGUUAGUUAGUUGCUCUUC
475-497
AD-53022.1
A-108471.1
UUAUUUGACUAUGCUGUUGGUUU
618-640
AD-53016.1
A-108469.1
UAUUUGACUAUGCUGUUGGUUUA
617-639
AD-53007.1
A-108419.1
UUCAAGUUUUGAGUUGAGUUCAA
404-426
AD-53148.1
A-108635.1
UUUGGUUGAUUUUAUAGAGUAUA
1373-1395
AD-53040.1
A-108477.1
UUCUUUUAUUUGACUAUGCUGUU
623-645
AD-53041.1
A-108493.1
AUGUUUUACAUUUCUUAUUUCAU
746-768
AD-53039.1
A-108461.1
AUUUUGAAUUAAGUUAGUUAGUU
482-504
AD-53139.1
A-108647.1
UUUAAAUUUGCCUCAGUUCAUUC
1429-1451
AD-53144.1
A-108649.1
UUUUAAAUUUGCCUCAGUUCAUU
1430-1452
AD-53142.1
A-108617.1
UAUUUACCAUUUAGGUUGUUUUC
1279-1301
AD-53108.1
A-108621.1
UAUAUUUACCAUUUAGGUUGUUU
1281-1303
AD-53079.1
A-108533.1
UAUUUGUAGUUCUCCCACGUUUC
940-962
AD-53133.1
A-108645.1
UUAAAUUUGCCUCAGUUCAUUCA
1428-1450
AD-53104.1
A-108557.1
UUGUCUUUCCAGUCUUCCAACUC
1051-1073
AD-53088.1
A-108583.1
AUUGCAUUGGGGACAUUGCCAGU
1147-1169
AD-53101.1
A-108509.1
AAUUAAUGUCCAUGGACUACCUG
884-906
AD-53000.1
A-108401.1
UUUUGACUUGUAGUUUAUAUGUA
357-379
AD-53112.1
A-108591.1
AAAUCUUUGUUUUCCGGGAUUGC
1165-1187
AD-53107.1
A-108605.1
UUGCUUUGUGAUCCCAAGUAGAA
1194-1216
AD-53121.1
A-108641.1
AAAUUUGCCUCAGUUCAUUCAAA
1426-1448
AD-53046.1
A-108479.1
UUUCUUUUAUUUGACUAUGCUGU
624-646
AD-53038.1
A-108445.1
UAGUUAGUUGCUCUUCUAAAUAU
468-490
AD-53140.1
A-108663.1
UAUUGCCUUUUAAAUUUGCCUCA
1437-1459
AD-52987.1
A-108381.1
UAAAAAGACUGAUCAAAUAUGUU
277-299
AD-53130.1
A-108597.1
AAAACACCAAAUCUUUGUUUUCC
1173-1195
AD-53106.1
A-108589.1
AAUCUUUGUUUUCCGGGAUUGCA
1164-1186
AD-53081.1
A-108565.1
UAUUCAAUAUAAUGUUUGUUGUC
1069-1091
AD-53118.1
A-108593.1
AACACCAAAUCUUUGUUUUCCGG
1171-1193
AD-53136.1
A-108599.1
UAGAAAACACCAAAUCUUUGUUU
1176-1198
AD-53127.1
A-108643.1
UAAAUUUGCCUCAGUUCAUUCAA
1427-1449
AD-53066.1
A-108513.1
AUGUUGAAUUAAUGUCCAUGGAC
890-912
AD-53013.1
A-108421.1
UUUCAAGUUUUGAGUUGAGUUCA
405-427
AD-52991.1
A-108351.1
UUUAAGACCAUGUCCCAACUGAA
203-225
AD-53099.1
A-108571.1
AAGAAUAUUCAAUAUAAUGUUUG
1074-1096
AD-52958.1
A-108387.1
UUCUUCUUUGAUUUCACUGGUUU
314-336
AD-53097.1
A-108539.1
UAUAUCUUCUCUAGGCCCAACCA
991-1013
AD-52966.1
A-108327.1
AUCUUGAUUUUGGCUCUGGAGAU
138-160
AD-53145.1
A-108665.1
UUAUUGCCUUUUAAAUUUGCCUC
1438-1460
AD-53113.1
A-108607.1
UUUGCUUUGUGAUCCCAAGUAGA
1195-1217
AD-52993.1
A-108383.1
UAGAUCAUAAAAAGACUGAUCAA
284-306
AD-53031.1
A-108427.1
UUUUUCUUCUAGGAGGCUUUCAA
422-444
AD-53017.1
A-108485.1
UUCUAUUUCUUUUAUUUGACUAU
629-651
AD-53143.1
A-108633.1
UUGGUUGAUUUUAUAGAGUAUAA
1372-1394
AD-53149.1
A-108651.1
UUUUUAAAUUUGCCUCAGUUCAU
1431-
1453_G21A
AD-53059.1
A-108495.1
AACUUGAGAGUUGCUGGGUCUGA
836-858
AD-53006.1
A-108403.1
UUUUUGACUUGUAGUUUAUAUGU
358-380
AD-53025.1
A-108425.1
UUUUCUUCUAGGAGGCUUUCAAG
421-443
AD-53085.1
A-108535.1
AACCAUAUUUGUAGUUCUCCCAC
945-967
AD-52984.1
A-108333.1
AUCGUCUAACAUAGCAAAUCUUG
155-177
AD-53023.1
A-108487.1
UAGAGAAAUUUCUGUGGGUUCUU
677-699
AD-53014.1
A-108437.1
UAUUUCACUUUUUGUUGAAGUAG
448-470
AD-53060.1
A-108511.1
UUGAAUUAAUGUCCAUGGACUAC
887-909
AD-53110.1
A-108653.1
UCUUUUAAAUUUGCCUCAGUUCA
1432-
1454_G21A
AD-52980.1
A-108363.1
AAUAUGUCAUUAAUUUGGCCCUU
244-266
AD-53109.1
A-108637.1
UUCUGAAUCUGUUGGAUGGAUCA
1400-1422
AD-53141.1
A-108601.1
AAGUAGAAAACACCAAAUCUUUG
1179-1201
AD-53126.1
A-108627.1
UAUAACCUUCCAUUUUGAGACUU
1354-1376
AD-53116.1
A-108655.1
UCCUUUUAAAUUUGCCUCAGUUC
1433-
1455_C21A
AD-52997.1
A-108353.1
AAGUCUUUAAGACCAUGUCCCAA
208-230
AD-53120.1
A-108625.1
UUGGUUUGUUAUAUUUACCAUUU
1290-1312
AD-53070.1
A-108577.1
UAGUUGGUUUCGUGAUUUCCCAA
1102-1124
AD-53028.1
A-108473.1
UUUAUUUGACUAUGCUGUUGGUU
619-641
AD-53146.1
A-108603.1
UUUGUGAUCCCAAGUAGAAAACA
1190-1212
AD-52982.1
A-108395.1
UUGUAGUUUAUAUGUAGUUCUUC
350-372
AD-53111.1
A-108669.1
ACAGAUUUUUACACAUACUCUGU
1913-1935
AD-53045.1
A-108463.1
UUGAUGCUAUUAUCUUGUUUUUC
556-578
AD-53123.1
A-108673.1
UUACAGAUUUUUACACAUACUCU
1915-1937
AD-53018.1
A-108407.1
UUUACCUCUUCAUUUUUGACUUG
370-392
AD-52956.1
A-108355.1
AAAGUCUUUAAGACCAUGUCCCA
209-231
AD-53134.1
A-108661.1
AUUGCCUUUUAAAUUUGCCUCAG
1436-1458
AD-52968.1
A-108359.1
UAUGGACAAAGUCUUUAAGACCA
216-238
AD-53122.1
A-108657.1
UGCCUUUUAAAUUUGCCUCAGUU
1434-1456
AD-53100.1
A-108587.1
AUCUUUGUUUUCCGGGAUUGCAU
1163-1185
AD-53128.1
A-108659.1
UUGCCUUUUAAAUUUGCCUCAGU
1435-1457
AD-53043.1
A-108431.1
UUCACUUUUUGUUGAAGUAGAAU
445-467
AD-53135.1
A-108677.1
UAUUACAGAUUUUUACACAUACU
1917-1939
AD-53094.1
A-108585.1
UUUGUUUUCCGGGAUUGCAUUGG
1160-1182
AD-53019.1
A-108423.1
UUCUUCUAGGAGGCUUUCAAGUU
419-441
AD-53129.1
A-108675.1
AUUACAGAUUUUUACACAUACUC
1916-1938
AD-53150.1
A-108667.1
AAGAUUUUUACACAUACUCUGUG
1912-
1934_G21U
AD-53117.1
A-108671.1
UACAGAUUUUUACACAUACUCUG
1914-1936
AD-52985.1
A-108349.1
UUAAGACCAUGUCCCAACUGAAG
202-224
AD-52962.1
A-108357.1
AUGGACAAAGUCUUUAAGACCAU
215-237
AD-52974.1
A-108361.1
UUAUGGACAAAGUCUUUAAGACC
217-239
AD-52979.1
A-108347.1
UAAGACCAUGUCCCAACUGAAGG
201-223
The symbol “x” indicates that the sequence contains a GalNAc conjugate.
TABLE 8
Modified sense and antisense strand sequences of ANGPTL3
GalNac-conjugated dsRNAs
Sense Sequence
Sense
(SEQ ID NOS 634-818, respectively,
Duplex ID
OligoName
in order of appearance)
AD-53063.1
A-108558.1
AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUfL96
AD-52965.1
A-108310.1
AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUfL96
AD-53030.1
A-108410.1
UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAfL96
AD-52953.1
A-108306.1
UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUfL96
AD-53001.1
A-108416.1
CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUfL96
AD-53080.1
A-108548.1
CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAfL96
AD-52971.1
A-108312.1
CfaAfuUfaAfgCfUfCfcUfuCfuUfuUfuAfL96
AD-53071.1
A-108498.1
AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUfL96
AD-53024.1
A-108408.1
GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAfL96
AD-52977.1
A-108314.1
AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUfL96
AD-53064.1
A-108574.1
CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUfL96
AD-53033.1
A-108458.1
AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAfL96
AD-52954.1
A-108322.1
UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUfL96
AD-53098.1
A-108554.1
CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAfL96
AD-53092.1
A-108552.1
CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUfL96
AD-53073.1
A-108530.1
GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAfL96
AD-53132.1
A-108628.1
AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAfL96
AD-53086.1
A-108550.1
UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUfL96
AD-52961.1
A-108340.1
CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAfL96
AD-52983.1
A-108316.1
AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUfL96
AD-53027.1
A-108456.1
AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAfL96
AD-52986.1
A-108364.1
GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUfL96
AD-52989.1
A-108318.1
UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUfL96
AD-52981.1
A-108378.1
AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96
AD-53077.1
A-108500.1
CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUfL96
AD-53095.1
A-108506.1
CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAfL96
AD-52970.1
A-108390.1
AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAfL96
AD-53015.1
A-108452.1
GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUfL96
AD-53147.1
A-108618.1
AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUfL96
AD-53103.1
A-108540.1
CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUfL96
AD-52969.1
A-108374.1
CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUfL96
AD-53075.1
A-108562.1
AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUfL96
AD-52994.1
A-108398.1
AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAfL96
AD-52960.1
A-108324.1
CfuAfgUfuAfuUfUfCfcUfcCfaGfaAfuUfL96
AD-53003.1
A-108448.1
AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAfL96
AD-52995.1
A-108320.1
UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUfL96
AD-53037.1
A-108428.1
CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAfL96
AD-53087.1
A-108566.1
AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUfL96
AD-53076.1
A-108578.1
GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUfL96
AD-52975.1
A-108376.1
AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUfL96
AD-53138.1
A-108630.1
UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAfL96
AD-53091.1
A-108536.1
GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUfL96
AD-53124.1
A-108594.1
GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUfL96
AD-53125.1
A-108610.1
AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAfL96
AD-53036.1
A-108412.1
GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAfL96
AD-53061.1
A-108526.1
GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUfL96
AD-53093.1
A-108568.1
AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUfL96
AD-53137.1
A-108614.1
UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUfL96
AD-52999.1
A-108384.1
AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUfL96
AD-53069.1
A-108560.1
GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAfL96
AD-53034.1
A-108474.1
CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAfL96
AD-52976.1
A-108392.1
CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAfL96
AD-52996.1
A-108336.1
UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAfL96
AD-53029.1
A-108488.1
AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUfL96
AD-53020.1
A-108438.1
CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUfL96
AD-53042.1
A-108414.1
UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAfL96
AD-53011.1
A-108482.1
CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAfL96
AD-52957.1
A-108370.1
CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUfL96
AD-53008.1
A-108434.1
UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUfL96
AD-53065.1
A-108496.1
GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUfL96
AD-53115.1
A-108638.1
UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUfL96
AD-53012.1
A-108404.1
UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUfL96
AD-53004.1
A-108464.1
AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAfL96
AD-53021.1
A-108454.1
CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAfL96
AD-52955.1
A-108338.1
GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAfL96
AD-53119.1
A-108608.1
AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAfL96
AD-52990.1
A-108334.1
UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAfL96
AD-52964.1
A-108388.1
AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAfL96
AD-52973.1
A-108344.1
GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUfL96
AD-53074.1
A-108546.1
AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAfL96
AD-53026.1
A-108440.1
UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUfL96
AD-53062.1
A-108542.1
CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAfL96
AD-53114.1
A-108622.1
CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAfL96
AD-53082.1
A-108580.1
GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAfL96
AD-53035.1
A-108490.1
CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUfL96
AD-52978.1
A-108330.1
AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAfL96
AD-53084.1
A-108518.1
AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAfL96
AD-52972.1
A-108328.1
CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUfL96
AD-53002.1
A-108432.1
CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAfL96
AD-53078.1
A-108516.1
GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUfL96
AD-53072.1
A-108514.1
GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAfL96
AD-53005.1
A-108480.1
GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUfL96
AD-53083.1
A-108502.1
CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAfL96
AD-53102.1
A-108524.1
AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAfL96
AD-53105.1
A-108572.1
AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUfL96
AD-53090.1
A-108520.1
UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUfL96
AD-53010.1
A-108466.1
GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUfL96
AD-52998.1
A-108368.1
UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAfL96
AD-52992.1
A-108366.1
AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAfL96
AD-53068.1
A-108544.1
GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAfL96
AD-53032.1
A-108442.1
AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAfL96
AD-52967.1
A-108342.1
CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAfL96
AD-53096.1
A-108522.1
UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUfL96
AD-53131.1
A-108612.1
GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAfL96
AD-52963.1
A-108372.1
UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUfL96
AD-53089.1
A-108504.1
UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAfL96
AD-53044.1
A-108446.1
UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAfL96
AD-52988.1
A-108396.1
UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAfL96
AD-53067.1
A-108528.1
GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAfL96
AD-53009.1
A-108450.1
AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUfL96
AD-53022.1
A-108470.1
AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAfL96
AD-53016.1
A-108468.1
AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAfL96
AD-53007.1
A-108418.1
GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAfL96
AD-53148.1
A-108634.1
UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAfL96
AD-53040.1
A-108476.1
CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAfL96
AD-53041.1
A-108492.1
GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUfL96
AD-53039.1
A-108460.1
CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUfL96
AD-53139.1
A-108646.1
AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAfL96
AD-53144.1
A-108648.1
UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAfL96
AD-53142.1
A-108616.1
AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAfL96
AD-53108.1
A-108620.1
AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAfL96
AD-53079.1
A-108532.1
AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAfL96
AD-53133.1
A-108644.1
AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAfL96
AD-53104.1
A-108556.1
GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAfL96
AD-53088.1
A-108582.1
UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUfL96
AD-53101.1
A-108508.1
GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUfL96
AD-53000.1
A-108400.1
CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAfL96
AD-53112.1
A-108590.1
AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUfL96
AD-53107.1
A-108604.1
CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAfL96
AD-53121.1
A-108640.1
UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUfL96
AD-53046.1
A-108478.1
AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAfL96
AD-53038.1
A-108444.1
AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAfL96
AD-53140.1
A-108662.1
AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAfL96
AD-52987.1
A-108380.1
CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAfL96
AD-53130.1
A-108596.1
AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUfL96
AD-53106.1
A-108588.1
CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUfL96
AD-53081.1
A-108564.1
CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAfL96
AD-53118.1
A-108592.1
GfgAfaAfaCfaAfAfGfaUfuUfgGfuGfuUfL96
AD-53136.1
A-108598.1
AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAfL96
AD-53127.1
A-108642.1
GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAfL96
AD-53066.1
A-108512.1
CfcAfuGfgAfcAfUfUfaAfuUfcAfaCfaUfL96
AD-53013.1
A-108420.1
AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAfL96
AD-52991.1
A-108350.1
CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAfL96
AD-53099.1
A-108570.1
AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUfL96
AD-52958.1
A-108386.1
AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAfL96
AD-53097.1
A-108538.1
GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAfL96
AD-52966.1
A-108326.1
CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUfL96
AD-53145.1
A-108664.1
GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAfL96
AD-53113.1
A-108606.1
UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAfL96
AD-52993.1
A-108382.1
GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAfL96
AD-53031.1
A-108426.1
GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAfL96
AD-53017.1
A-108484.1
AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAfL96
AD-53143.1
A-108632.1
AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAfL96
AD-53149.1
A-108650.1
GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAfL96
AD-53059.1
A-108494.1
AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUfL96
AD-53006.1
A-108402.1
AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAfL96
AD-53025.1
A-108424.1
UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAfL96
AD-53085.1
A-108534.1
GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUfL96
AD-52984.1
A-108332.1
AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUfL96
AD-53023.1
A-108486.1
GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAfL96
AD-53014.1
A-108436.1
AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAfL96
AD-53060.1
A-108510.1
AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAfL96
AD-53110.1
A-108652.1
AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAfL96
AD-52980.1
A-108362.1
GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUfL96
AD-53109.1
A-108636.1
AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAfL96
AD-53141.1
A-108600.1
AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUfL96
AD-53126.1
A-108626.1
GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAfL96
AD-53116.1
A-108654.1
AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAfL96
AD-52997.1
A-108352.1
GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUfL96
AD-53120.1
A-108624.1
AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAfL96
AD-53070.1
A-108576.1
GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAfL96
AD-53028.1
A-108472.1
CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAfL96
AD-53146.1
A-108602.1
UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAfL96
AD-52982.1
A-108394.1
AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAfL96
AD-53111.1
A-108668.1
AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUfL96
AD-53045.1
A-108462.1
AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAfL96
AD-53123.1
A-108672.1
AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAfL96
AD-53018.1
A-108406.1
AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAfL96
AD-52956.1
A-108354.1
GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUfL96
AD-53134.1
A-108660.1
GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUfL96
AD-52968.1
A-108358.1
GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAfL96
AD-53122.1
A-108656.1
CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAfL96
AD-53100.1
A-108586.1
GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUfL96
AD-53128.1
A-108658.1
UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAfL96
AD-53043.1
A-108430.1
UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAfL96
AD-53135.1
A-108676.1
UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAfL96
AD-53094.1
A-108584.1
AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAfL96
AD-53019.1
A-108422.1
CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAfL96
AD-53129.1
A-108674.1
GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUfL96
AD-53150.1
A-108666.1
CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUfL96
AD-53117.1
A-108670.1
GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAfL96
AD-52985.1
A-108348.1
UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAfL96
AD-52962.1
A-108356.1
GfgUfcUfuAfaAfGfAfcUfuUfgUfcCfaUfL96
AD-52974.1
A-108360.1
UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAfL96
AD-52979.1
A-108346.1
UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAfL96
Antisense Sequence
Antisense
(SEQ ID NOS 819-1003, respectively,
Duplex ID
OligoName
in order of appearance)
AD-53063.1
A-108559.1
aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc
AD-52965.1
A-108311.1
aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa
AD-53030.1
A-108411.1
uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa
AD-52953.1
A-108307.1
aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc
AD-53001.1
A-108417.1
aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg
AD-53080.1
A-108549.1
uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa
AD-52971.1
A-108313.1
uAfaAfaAfgAfaGfgagCfuUfaAfuUfgsUfsg
AD-53071.1
A-108499.1
aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu
AD-53024.1
A-108409.1
uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu
AD-52977.1
A-108315.1
aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu
AD-53064.1
A-108575.1
aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu
AD-53033.1
A-108459.1
uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg
AD-52954.1
A-108323.1
aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa
AD-53098.1
A-108555.1
uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa
AD-53092.1
A-108553.1
aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg
AD-53073.1
A-108531.1
uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa
AD-53132.1
A-108629.1
uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu
AD-53086.1
A-108551.1
aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu
AD-52961.1
A-108341.1
uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa
AD-52983.1
A-108317.1
aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg
AD-53027.1
A-108457.1
uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc
AD-52986.1
A-108365.1
aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu
AD-52989.1
A-108319.1
aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg
AD-52981.1
A-108379.1
aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg
AD-53077.1
A-108501.1
aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc
AD-53095.1
A-108507.1
uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu
AD-52970.1
A-108391.1
uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc
AD-53015.1
A-108453.1
aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu
AD-53147.1
A-108619.1
aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu
AD-53103.1
A-108541.1
aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc
AD-52969.1
A-108375.1
aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg
AD-53075.1
A-108563.1
aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu
AD-52994.1
A-108399.1
uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg
AD-52960.1
A-108325.1
aAfuUfcUfgGfaGfgaaAfuAfaCfuAfgsAfsg
AD-53003.1
A-108449.1
uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu
AD-52995.1
A-108321.1
aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa
AD-53037.1
A-108429.1
uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc
AD-53087.1
A-108567.1
aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu
AD-53076.1
A-108579.1
aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa
AD-52975.1
A-108377.1
aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa
AD-53138.1
A-108631.1
uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu
AD-53091.1
A-108537.1
aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa
AD-53124.1
A-108595.1
aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg
AD-53125.1
A-108611.1
uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa
AD-53036.1
A-108413.1
uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu
AD-53061.1
A-108527.1
aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa
AD-53093.1
A-108569.1
aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg
AD-53137.1
A-108615.1
aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu
AD-52999.1
A-108385.1
aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa
AD-53069.1
A-108561.1
uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu
AD-53034.1
A-108475.1
uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu
AD-52976.1
A-108393.1
uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu
AD-52996.1
A-108337.1
uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa
AD-53029.1
A-108489.1
aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu
AD-53020.1
A-108439.1
aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa
AD-53042.1
A-108415.1
uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa
AD-53011.1
A-108483.1
uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu
AD-52957.1
A-108371.1
aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa
AD-53008.1
A-108435.1
aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa
AD-53065.1
A-108497.1
aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg
AD-53115.1
A-108639.1
aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg
AD-53012.1
A-108405.1
aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg
AD-53004.1
A-108465.1
uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu
AD-53021.1
A-108455.1
uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu
AD-52955.1
A-108339.1
uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa
AD-53119.1
A-108609.1
uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg
AD-52990.1
A-108335.1
uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu
AD-52964.1
A-108389.1
uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc
AD-52973.1
A-108345.1
aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu
AD-53074.1
A-108547.1
uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu
AD-53026.1
A-108441.1
aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu
AD-53062.1
A-108543.1
uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc
AD-53114.1
A-108623.1
uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu
AD-53082.1
A-108581.1
uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc
AD-53035.1
A-108491.1
aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu
AD-52978.1
A-108331.1
uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg
AD-53084.1
A-108519.1
uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc
AD-52972.1
A-108329.1
aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg
AD-53002.1
A-108433.1
uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa
AD-53078.1
A-108517.1
aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa
AD-53072.1
A-108515.1
uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu
AD-53005.1
A-108481.1
aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg
AD-53083.1
A-108503.1
uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu
AD-53102.1
A-108525.1
uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu
AD-53105.1
A-108573.1
aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu
AD-53090.1
A-108521.1
aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg
AD-53010.1
A-108467.1
aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu
AD-52998.1
A-108369.1
uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu
AD-52992.1
A-108367.1
uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg
AD-53068.1
A-108545.1
uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc
AD-53032.1
A-108443.1
uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc
AD-52967.1
A-108343.1
uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc
AD-53096.1
A-108523.1
aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu
AD-53131.1
A-108613.1
uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc
AD-52963.1
A-108373.1
aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu
AD-53089.1
A-108505.1
uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa
AD-53044.1
A-108447.1
uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa
AD-52988.1
A-108397.1
uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu
AD-53067.1
A-108529.1
uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu
AD-53009.1
A-108451.1
aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc
AD-53022.1
A-108471.1
uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu
AD-53016.1
A-108469.1
uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa
AD-53007.1
A-108419.1
uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa
AD-53148.1
A-108635.1
uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa
AD-53040.1
A-108477.1
uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu
AD-53041.1
A-108493.1
aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu
AD-53039.1
A-108461.1
aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu
AD-53139.1
A-108647.1
uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc
AD-53144.1
A-108649.1
uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu
AD-53142.1
A-108617.1
uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc
AD-53108.1
A-108621.1
uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu
AD-53079.1
A-108533.1
uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc
AD-53133.1
A-108645.1
uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa
AD-53104.1
A-108557.1
uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc
AD-53088.1
A-108583.1
aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu
AD-53101.1
A-108509.1
aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg
AD-53000.1
A-108401.1
uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa
AD-53112.1
A-108591.1
aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc
AD-53107.1
A-108605.1
uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa
AD-53121.1
A-108641.1
aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa
AD-53046.1
A-108479.1
uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu
AD-53038.1
A-108445.1
uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu
AD-53140.1
A-108663.1
uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa
AD-52987.1
A-108381.1
uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu
AD-53130.1
A-108597.1
aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc
AD-53106.1
A-108589.1
aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa
AD-53081.1
A-108565.1
uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc
AD-53118.1
A-108593.1
aAfcAfcCfaAfaUfcuuUfgUfuUfuCfcsGfsg
AD-53136.1
A-108599.1
uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu
AD-53127.1
A-108643.1
uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa
AD-53066.1
A-108513.1
aUfgUfuGfaAfuUfaauGfuCfcAfuGfgsAfsc
AD-53013.1
A-108421.1
uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa
AD-52991.1
A-108351.1
uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa
AD-53099.1
A-108571.1
aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg
AD-52958.1
A-108387.1
uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu
AD-53097.1
A-108539.1
uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa
AD-52966.1
A-108327.1
aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu
AD-53145.1
A-108665.1
uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc
AD-53113.1
A-108607.1
uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa
AD-52993.1
A-108383.1
uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa
AD-53031.1
A-108427.1
uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa
AD-53017.1
A-108485.1
uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu
AD-53143.1
A-108633.1
uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa
AD-53149.1
A-108651.1
uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu
AD-53059.1
A-108495.1
aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa
AD-53006.1
A-108403.1
uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu
AD-53025.1
A-108425.1
uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg
AD-53085.1
A-108535.1
aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc
AD-52984.1
A-108333.1
aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg
AD-53023.1
A-108487.1
uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu
AD-53014.1
A-108437.1
uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg
AD-53060.1
A-108511.1
uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc
AD-53110.1
A-108653.1
uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa
AD-52980.1
A-108363.1
aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu
AD-53109.1
A-108637.1
uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa
AD-53141.1
A-108601.1
aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg
AD-53126.1
A-108627.1
uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu
AD-53116.1
A-108655.1
uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc
AD-52997.1
A-108353.1
aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa
AD-53120.1
A-108625.1
uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu
AD-53070.1
A-108577.1
uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa
AD-53028.1
A-108473.1
uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu
AD-53146.1
A-108603.1
uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa
AD-52982.1
A-108395.1
uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc
AD-53111.1
A-108669.1
aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu
AD-53045.1
A-108463.1
uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc
AD-53123.1
A-108673.1
uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu
AD-53018.1
A-108407.1
uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg
AD-52956.1
A-108355.1
aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa
AD-53134.1
A-108661.1
aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg
AD-52968.1
A-108359.1
uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa
AD-53122.1
A-108657.1
uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu
AD-53100.1
A-108587.1
aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu
AD-53128.1
A-108659.1
uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu
AD-53043.1
A-108431.1
uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu
AD-53135.1
A-108677.1
uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu
AD-53094.1
A-108585.1
uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg
AD-53019.1
A-108423.1
uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu
AD-53129.1
A-108675.1
aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc
AD-53150.1
A-108667.1
aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg
AD-53117.1
A-108671.1
uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg
AD-52985.1
A-108349.1
uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg
AD-52962.1
A-108357.1
aUfgGfaCfaAfaGfucuUfuAfaGfaCfcsAfsu
AD-52974.1
A-108361.1
uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc
AD-52979.1
A-108347.1
uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; Nf (e.g., Af) is a 2′-fluoro nucleotide; s is a phosphothiorate linkage; L96 indicates a GalNAc ligand.
TABLE 9
Unmodified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences arc the same as the sequences
listed in Table 7 except that they do not
contain GalNal conjugation.
Sense
Antisense
Sequence
Sequence
(SEQ ID NOS
(SEQ ID NOS
1004-1184,
1185-1365,
respectively,
respectively,
Duplex
Sense
in order of
Antisense
in order of
Position in
Name
OligoName
appearance)
OligoName
appearance)
NM_014495.2
AD-52637.1
A-108817.1
UCACAAUUAAGCUCCUUCUUU
A-108307.2
AAAGAAGGAGCUUAAUUGUGAAC
54-76
AD-52638.1
A-108825.1
UUAUUGUUCCUCUAGUUAUUU
A-108323.2
AAAUAACUAGAGGAACAAUAAAA
75-97
AD-52639.1
A-108833.1
GCUAUGUUAGACGAUGUAAAA
A-108339.2
UUUUACAUCGUCUAACAUAGCAA
161-183
AD-52640.1
A-108841.1
GGACAUGGUCUUAAAGACUUU
A-108355.2
AAAGUCUUUAAGACCAUGUCCCA
209-231
AD-52641.1
A-108849.1
CAAAAACUCAACAUAUUUGAU
A-108371.2
AUCAAAUAUGUUGAGUUUUUGAA
266-288
AD-52642.1
A-108857.1
ACCAGUGAAAUCAAAGAAGAA
A-108387.2
UUCUUCUUUGAUUUCACUGGUUU
314-336
AD-52643.1
A-108818.1
CACAAUUAAGCUCCUUCUUUU
A-108309.2
AAAAGAAGGAGCUUAAUUGUGAA
55-77
AD-52645.1
A-108834.1
CUAUGUUAGACGAUGUAAAAA
A-108341.2
UUUUUACAUCGUCUAACAUAGCA
162-184
AD-52647.1
A-108850.1
UCAACAUAUUUGAUCAGUCUU
A-108373.2
AAGACUGAUCAAAUAUGUUGAGU
273-295
AD-52648.1
A-108858.1
AACUGAGAAGAACUACAUAUA
A-108389.2
UAUAUGUAGUUCUUCUCAGUUCC
342-364
AD-52649.1
A-108819.1
ACAAUUAAGCUCCUUCUUUUU
A-108311.2
AAAAAGAAGGAGCUUAAUUGUGA
56-78
AD-52650.1
A-108827.1
CUCCAGAGCCAAAAUCAAGAU
A-108327.2
AUCUUGAUUUUGGCUCUGGAGAU
138-160
AD-52651.1
A-108835.1
CGAUGUAAAAAUUUUAGCCAA
A-108343.2
UUGGCUAAAAUUUUUACAUCGUC
172-194
AD-52652.1
A-108843.1
GUCUUAAAGACUUUGUCCAUA
A-108359.2
UAUGGACAAAGUCUUUAAGACCA
216-238
AD-52653.1
A-108851.1
CAACAUAUUUGAUCAGUCUUU
A-108375.2
AAAGACUGAUCAAAUAUGUUGAG
274-296
AD-52654.1
A-108859.1
ACUGAGAAGAACUACAUAUAA
A-108391.2
UUAUAUGUAGUUCUUCUCAGUUC
343-365
AD-52656.1
A-108828.1
CCAGAGCCAAAAUCAAGAUUU
A-108329.2
AAAUCUUGAUUUUGGCUCUGGAG
140-162
AD-52657.1
A-108836.1
GAUGUAAAAAUUUUAGCCAAU
A-108345.2
AUUGGCUAAAAUUUUUACAUCGU
173-195
AD-52658.1
A-108844.1
UCUUAAAGACUUUGUCCAUAA
A-108361.2
UUAUGGACAAAGUCUUUAAGACC
217-239
AD-52659.1
A-108852.1
AACAUAUUUGAUCAGUCUUUU
A-108377.2
AAAAGACUGAUCAAAUAUGUUGA
275-297
AD-52660.1
A-108860.1
CUGAGAAGAACUACAUAUAAA
A-108393.2
UUUAUAUGUAGUUCUUCUCAGUU
344-366
AD-52661.1
A-108821.1
AAUUAAGCUCCUUCUUUUUAU
A-108315.2
AUAAAAAGAAGGAGCUUAAUUGU
58-80
AD-52662.1
A-108829.1
AAAUCAAGAUUUGCUAUGUUA
A-108331.2
UAACAUAGCAAAUCUUGAUUUUG
149-171
AD-52663.1
A-108837.1
UUCAGUUGGGACAUGGUCUUA
A-108347.2
UAAGACCAUGUCCCAACUGAAGG
201-223
AD-52664.1
A-108845.1
GGGCCAAAUUAAUGACAUAUU
A-108363.2
AAUAUGUCAUUAAUUUGGCCCUU
244-266
AD-52665.1
A-108853.1
ACAUAUUUGAUCAGUCUUUUU
A-108379.2
AAAAAGACUGAUCAAAUAUGUUG
276-298
AD-52666.1
A-108861.1
AGAACUACAUAUAAACUACAA
A-108395.2
UUGUAGUUUAUAUGUAGUUCUUC
350-372
AD-52667.1
A-108822.1
AUUAAGCUCCUUCUUUUUAUU
A-108317.2
AAUAAAAAGAAGGAGCUUAAUUG
59-81
AD-52668.1
A-108830.1
AGAUUUGCUAUGUUAGACGAU
A-108333.2
AUCGUCUAACAUAGCAAAUCUUG
155-177
AD-52669.1
A-108838.1
UCAGUUGGGACAUGGUCUUAA
A-108349.2
UUAAGACCAUGUCCCAACUGAAG
202-224
AD-52670.1
A-108846.1
GGCCAAAUUAAUGACAUAUUU
A-108365.2
AAAUAUGUCAUUAAUUUGGCCCU
245-267
AD-52671.1
A-108854.1
CAUAUUUGAUCAGUCUUUUUA
A-108381.2
UAAAAAGACUGAUCAAAUAUGUU
277-299
AD-52672.1
A-108862.1
UACAUAUAAACUACAAGUCAA
A-108397.2
UUGACUUGUAGUUUAUAUGUAGU
355-377
AD-52673.1
A-108823.1
UUUUAUUGUUCCUCUAGUUAU
A-108319.2
AUAACUAGAGGAACAAUAAAAAG
73-95
AD-52674.1
A-108831.1
UUGCUAUGUUAGACGAUGUAA
A-108335.2
UUACAUCGUCUAACAUAGCAAAU
159-181
AD-52675.1
A-108839.1
CAGUUGGGACAUGGUCUUAAA
A-108351.2
UUUAAGACCAUGUCCCAACUGAA
203-225
AD-52676.1
A-108847.1
AAAUUAAUGACAUAUUUCAAA
A-108367.2
UUUGAAAUAUGUCAUUAAUUUGG
249-271
AD-52677.1
A-108855.1
GAUCAGUCUUUUUAUGAUCUA
A-108383.2
UAGAUCAUAAAAAGACUGAUCAA
284-306
AD-52678.1
A-108863.1
ACAUAUAAACUACAAGUCAAA
A-108399.2
UUUGACUUGUAGUUUAUAUGUAG
356-378
AD-52679.1
A-108824.1
UUUAUUGUUCCUCUAGUUAUU
A-108321.2
AAUAACUAGAGGAACAAUAAAAA
74-96
AD-52680.1
A-108832.1
UGCUAUGUUAGACGAUGUAAA
A-108337.2
UUUACAUCGUCUAACAUAGCAAA
160-182
AD-52681.1
A-108840.1
GGGACAUGGUCUUAAAGACUU
A-108353.2
AAGUCUUUAAGACCAUGUCCCAA
208-230
AD-52682.1
A-108848.1
UGACAUAUUUCAAAAACUCAA
A-108369.2
UUGAGUUUUUGAAAUAUGUCAUU
256-278
AD-52683.1
A-108856.1
AUCAGUCUUUUUAUGAUCUAU
A-108385.2
AUAGAUCAUAAAAAGACUGAUCA
285-307
AD-52684.1
A-108864.1
CAUAUAAACUACAAGUCAAAA
A-108401.2
UUUUGACUUGUAGUUUAUAUGUA
357-379
AD-52685.1
A-108872.1
CUUGAACUCAACUCAAAACUU
A-108417.2
AAGUUUUGAGUUGAGUUCAAGUG
401-423
AD-52686.1
A-108880.1
CUACUUCAACAAAAAGUGAAA
A-108433.2
UUUCACUUUUUGUUGAAGUAGAA
446-468
AD-52687.1
A-108888.1
AAGAGCAACUAACUAACUUAA
A-108449.2
UUAAGUUAGUUAGUUGCUCUUCU
474-496
AD-52688.1
A-108896.1
AAACAAGAUAAUAGCAUCAAA
A-108465.2
UUUGAUGCUAUUAUCUUGUUUUU
557-579
AD-52689.1
A-108904.1
GCAUAGUCAAAUAAAAGAAAU
A-108481.2
AUUUCUUUUAUUUGACUAUGCUG
625-647
AD-52690.1
A-108865.1
AUAUAAACUACAAGUCAAAAA
A-108403.2
UUUUUGACUUGUAGUUUAUAUGU
358-380
AD-52691.1
A-108873.1
GAACUCAACUCAAAACUUGAA
A-108419.2
UUCAAGUUUUGAGUUGAGUUCAA
404-426
AD-52692.1
A-108881.1
UACUUCAACAAAAAGUGAAAU
A-108435.2
AUUUCACUUUUUGUUGAAGUAGA
447-469
AD-52693.1
A-108889.1
AGAGCAACUAACUAACUUAAU
A-108451.2
AUUAAGUUAGUUAGUUGCUCUUC
475-497
AD-52694.1
A-108897.1
GAUAAUAGCAUCAAAGACCUU
A-108467.2
AAGGUCUUUGAUGCUAUUAUCUU
563-585
AD-52695.1
A-108905.1
CAUAGUCAAAUAAAAGAAAUA
A-108483.2
UAUUUCUUUUAUUUGACUAUGCU
626-648
AD-52696.1
A-108866.1
UAUAAACUACAAGUCAAAAAU
A-108405.2
AUUUUUGACUUGUAGUUUAUAUG
359-381
AD-52697.1
A-108874.1
AACUCAACUCAAAACUUGAAA
A-108421.2
UUUCAAGUUUUGAGUUGAGUUCA
405-427
AD-52698.1
A-108882.1
ACUUCAACAAAAAGUGAAAUA
A-108437.2
UAUUUCACUUUUUGUUGAAGUAG
448-470
AD-52699.1
A-108890.1
GAGCAACUAACUAACUUAAUU
A-108453.2
AAUUAAGUUAGUUAGUUGCUCUU
476-498
AD-52700.1
A-108898.1
AACCAACAGCAUAGUCAAAUA
A-108469.2
UAUUUGACUAUGCUGUUGGUUUA
617-639
AD-52701.1
A-108906.1
AGUCAAAUAAAAGAAAUAGAA
A-108485.2
UUCUAUUUCUUUUAUUUGACUAU
629-651
AD-52702.1
A-108867.1
AGUCAAAAAUGAAGAGGUAAA
A-108407.2
UUUACCUCUUCAUUUUUGACUUG
370-392
AD-52703.1
A-108875.1
CUUGAAAGCCUCCUAGAAGAA
A-108423.2
UUCUUCUAGGAGGCUUUCAAGUU
419-441
AD-52704.1
A-108883.1
CUUCAACAAAAAGUGAAAUAU
A-108439.2
AUAUUUCACUUUUUGUUGAAGUA
449-471
AD-52705.1
A-108891.1
CAACUAACUAACUUAAUUCAA
A-108455.2
UUGAAUUAAGUUAGUUAGUUGCU
479-501
AD-52706.1
A-108899.1
ACCAACAGCAUAGUCAAAUAA
A-108471.2
UUAUUUGACUAUGCUGUUGGUUU
618-640
AD-52707.1
A-108907.1
GAACCCACAGAAAUUUCUCUA
A-108487.2
UAGAGAAAUUUCUGUGGGUUCUU
677-699
AD-52708.1
A-108868.1
GAAUAUGUCACUUGAACUCAA
A-108409.2
UUGAGUUCAAGUGACAUAUUCUU
391-413
AD-52709.1
A-108876.1
UGAAAGCCUCCUAGAAGAAAA
A-108425.2
UUUUCUUCUAGGAGGCUUUCAAG
421-443
AD-52710.1
A-108884.1
UUCAACAAAAAGUGAAAUAUU
A-108441.2
AAUAUUUCACUUUUUGUUGAAGU
450-472
AD-52711.1
A-108892.1
AACUAACUAACUUAAUUCAAA
A-108457.2
UUUGAAUUAAGUUAGUUAGUUGC
480-502
AD-52712.1
A-108900.1
CCAACAGCAUAGUCAAAUAAA
A-108473.2
UUUAUUUGACUAUGCUGUUGGUU
619-641
AD-52713.1
A-108908.1
AACCCACAGAAAUUUCUCUAU
A-108489.2
AUAGAGAAAUUUCUGUGGGUUCU
678-700
AD-52714.1
A-108869.1
UGUCACUUGAACUCAACUCAA
A-108411.2
UUGAGUUGAGUUCAAGUGACAUA
396-418
AD-52715.1
A-108877.1
GAAAGCCUCCUAGAAGAAAAA
A-108427.2
UUUUUCUUCUAGGAGGCUUUCAA
422-444
AD-52716.1
A-108885.1
AAUAUUUAGAAGAGCAACUAA
A-108443.2
UUAGUUGCUCUUCUAAAUAUUUC
465-487
AD-52717.1
A-108893.1
ACUAACUAACUUAAUUCAAAA
A-108459.2
UUUUGAAUUAAGUUAGUUAGUUG
481-503
AD-52718.1
A-108901.1
CAACAGCAUAGUCAAAUAAAA
A-108475.2
UUUUAUUUGACUAUGCUGUUGGU
620-642
AD-52719.1
A-108909.1
CCACAGAAAUUUCUCUAUCUU
A-108491.2
AAGAUAGAGAAAUUUCUGUGGGU
681-703
AD-52720.1
A-108870.1
GUCACUUGAACUCAACUCAAA
A-108413.2
UUUGAGUUGAGUUCAAGUGACAU
397-419
AD-52721.1
A-108878.1
CUCCUAGAAGAAAAAAUUCUA
A-108429.2
UAGAAUUUUUUCUUCUAGGAGGC
428-450
AD-52722.1
A-108886.1
AUUUAGAAGAGCAACUAACUA
A-108445.2
UAGUUAGUUGCUCUUCUAAAUAU
468-490
AD-52723.1
A-108894.1
CUAACUAACUUAAUUCAAAAU
A-108461.2
AUUUUGAAUUAAGUUAGUUAGUU
482-504
AD-52724.1
A-108902.1
CAGCAUAGUCAAAUAAAAGAA
A-108477.2
UUCUUUUAUUUGACUAUGCUGUU
623-645
AD-52725.1
A-108910.1
GAAAUAAGAAAUGUAAAACAU
A-108493.2
AUGUUUUACAUUUCUUAUUUCAU
746-768
AD-52726.1
A-108871.1
UCACUUGAACUCAACUCAAAA
A-108415.2
UUUUGAGUUGAGUUCAAGUGACA
398-420
AD-52727.1
A-108879.1
UCUACUUCAACAAAAAGUGAA
A-108431.2
UUCACUUUUUGUUGAAGUAGAAU
445-467
AD-52728.1
A-108887.1
UUUAGAAGAGCAACUAACUAA
A-108447.2
UUAGUUAGUUGCUCUUCUAAAUA
469-491
AD-52729.1
A-108895.1
AAAACAAGAUAAUAGCAUCAA
A-108463.2
UUGAUGCUAUUAUCUUGUUUUUC
556-578
AD-52730.1
A-108903.1
AGCAUAGUCAAAUAAAAGAAA
A-108479.2
UUUCUUUUAUUUGACUAUGCUGU
624-646
AD-52731.1
A-108958.1
AGACCCAGCAACUCUCAAGUU
A-108495.2
AACUUGAGAGUUGCUGGGUCUGA
836-858
AD-52732.1
A-108966.1
AGUCCAUGGACAUUAAUUCAA
A-108511.2
UUGAAUUAAUGUCCAUGGACUAC
887-909
AD-52733.1
A-108974.1
GAUGGAUCACAAAACUUCAAU
A-108527.2
AUUGAAGUUUUGUGAUCCAUCUA
917-939
AD-52734.1
A-108982.1
CUAGAGAAGAUAUACUCCAUA
A-108543.2
UAUGGAGUAUAUCUUCUCUAGGC
998-1020
AD-52735.1
A-108990.1
AAAGACAACAAACAUUAUAUU
A-108559.2
AAUAUAAUGUUUGUUGUCUUUCC
1064-1086
AD-52736.1
A-108998.1
CAUUAUAUUGAAUAUUCUUUU
A-108575.2
AAAAGAAUAUUCAAUAUAAUGUU
1076-1098
AD-52737.1
A-108959.1
GACCCAGCAACUCUCAAGUUU
A-108497.2
AAACUUGAGAGUUGCUGGGUCUG
837-859
AD-52739.1
A-108975.1
GGAUCACAAAACUUCAAUGAA
A-108529.2
UUCAUUGAAGUUUUGUGAUCCAU
920-942
AD-52740.1
A-108983.1
GAAGAUAUACUCCAUAGUGAA
A-108545.2
UUCACUAUGGAGUAUAUCUUCUC
1003-1025
AD-52741.1
A-108991.1
GACAACAAACAUUAUAUUGAA
A-108561.2
UUCAAUAUAAUGUUUGUUGUCUU
1067-1089
AD-52742.1
A-108999.1
GGGAAAUCACGAAACCAACUA
A-108577.2
UAGUUGGUUUCGUGAUUUCCCAA
1102-1124
AD-52743.1
A-108960.1
ACCCAGCAACUCUCAAGUUUU
A-108499.2
AAAACUUGAGAGUUGCUGGGUCU
838-860
AD-52744.1
A-108968.1
GGACAUUAAUUCAACAUCGAA
A-108515.2
UUCGAUGUUGAAUUAAUGUCCAU
894-916
AD-52745.1
A-108976.1
GAUCACAAAACUUCAAUGAAA
A-108531.2
UUUCAUUGAAGUUUUGUGAUCCA
921-943
AD-52746.1
A-108984.1
ACUCCAUAGUGAAGCAAUCUA
A-108547.2
UAGAUUGCUUCACUAUGGAGUAU
1011-1033
AD-52747.1
A-108992.1
ACAACAAACAUUAUAUUGAAU
A-108563.2
AUUCAAUAUAAUGUUUGUUGUCU
1068-1090
AD-52748.1
A-109000.1
GGAAAUCACGAAACCAACUAU
A-108579.2
AUAGUUGGUUUCGUGAUUUCCCA
1103-1125
AD-52749.1
A-108961.1
CCCAGCAACUCUCAAGUUUUU
A-108501.2
AAAAACUUGAGAGUUGCUGGGUC
839-861
AD-52750.1
A-108969.1
GACAUUAAUUCAACAUCGAAU
A-108517.2
AUUCGAUGUUGAAUUAAUGUCCA
895-917
AD-52751.1
A-108977.1
AACGUGGGAGAACUACAAAUA
A-108533.2
UAUUUGUAGUUCUCCCACGUUUC
940-962
AD-52752.1
A-108985.1
CUCCAUAGUGAAGCAAUCUAA
A-108549.2
UUAGAUUGCUUCACUAUGGAGUA
1012-1034
AD-52753.1
A-108993.1
CAACAAACAUUAUAUUGAAUA
A-108565.2
UAUUCAAUAUAAUGUUUGUUGUC
1069-1091
AD-52754.1
A-109001.1
GAAAUCACGAAACCAACUAUA
A-108581.2
UAUAGUUGGUUUCGUGAUUUCCC
1104-1126
AD-52755.1
A-108962.1
CUCUCAAGUUUUUCAUGUCUA
A-108503.2
UAGACAUGAAAAACUUGAGAGUU
847-869
AD-52756.1
A-108970.1
ACAUUAAUUCAACAUCGAAUA
A-108519.2
UAUUCGAUGUUGAAUUAAUGUCC
896-918
AD-52757.1
A-108978.1
GGGAGAACUACAAAUAUGGUU
A-108535.2
AACCAUAUUUGUAGUUCUCCCAC
945-967
AD-52758.1
A-108986.1
UCCAUAGUGAAGCAAUCUAAU
A-108551.2
AUUAGAUUGCUUCACUAUGGAGU
1013-1035
AD-52759.1
A-108994.1
AACAAACAUUAUAUUGAAUAU
A-108567.2
AUAUUCAAUAUAAUGUUUGUUGU
1070-1092
AD-52760.1
A-109002.1
UGGCAAUGUCCCCAAUGCAAU
A-108583.2
AUUGCAUUGGGGACAUUGCCAGU
1147-1169
AD-52761.1
A-108963.1
UCAGGUAGUCCAUGGACAUUA
A-108505.2
UAAUGUCCAUGGACUACCUGAUA
881-903
AD-52762.1
A-108971.1
UUAAUUCAACAUCGAAUAGAU
A-108521.2
AUCUAUUCGAUGUUGAAUUAAUG
899-921
AD-52763.1
A-108979.1
GGAGAACUACAAAUAUGGUUU
A-108537.2
AAACCAUAUUUGUAGUUCUCCCA
946-968
AD-52764.1
A-108987.1
CCAUAGUGAAGCAAUCUAAUU
A-108553.2
AAUUAGAUUGCUUCACUAUGGAG
1014-1036
AD-52765.1
A-108995.1
ACAAACAUUAUAUUGAAUAUU
A-108569.2
AAUAUUCAAUAUAAUGUUUGUUG
1071-1093
AD-52766.1
A-109003.1
AAUGCAAUCCCGGAAAACAAA
A-108585.2
UUUGUUUUCCGGGAUUGCAUUGG
1160-1182
AD-52767.1
A-108964.1
CAGGUAGUCCAUGGACAUUAA
A-108507.2
UUAAUGUCCAUGGACUACCUGAU
882-904
AD-52768.1
A-108972.1
UUCAACAUCGAAUAGAUGGAU
A-108523.2
AUCCAUCUAUUCGAUGUUGAAUU
903-925
AD-52769.1
A-108980.1
GUUGGGCCUAGAGAAGAUAUA
A-108539.2
UAUAUCUUCUCUAGGCCCAACCA
991-1013
AD-52770.1
A-108988.1
CAUAGUGAAGCAAUCUAAUUA
A-108555.2
UAAUUAGAUUGCUUCACUAUGGA
1015-1037
AD-52771.1
A-108996.1
AACAUUAUAUUGAAUAUUCUU
A-108571.2
AAGAAUAUUCAAUAUAAUGUUUG
1074-1096
AD-52772.1
A-109004.1
GCAAUCCCGGAAAACAAAGAU
A-108587.2
AUCUUUGUUUUCCGGGAUUGCAU
1163-1185
AD-52773.1
A-108965.1
GGUAGUCCAUGGACAUUAAUU
A-108509.2
AAUUAAUGUCCAUGGACUACCUG
884-906
AD-52774.1
A-108973.1
AUCGAAUAGAUGGAUCACAAA
A-108525.2
UUUGUGAUCCAUCUAUUCGAUGU
909-931
AD-52775.1
A-108981.1
CCUAGAGAAGAUAUACUCCAU
A-108541.2
AUGGAGUAUAUCUUCUCUAGGCC
997-1019
AD-52776.1
A-108989.1
GUUGGAAGACUGGAAAGACAA
A-108557.2
UUGUCUUUCCAGUCUUCCAACUC
1051-1073
AD-52777.1
A-108997.1
ACAUUAUAUUGAAUAUUCUUU
A-108573.2
AAAGAAUAUUCAAUAUAAUGUUU
1075-1097
AD-52778.1
A-109005.1
CAAUCCCGGAAAACAAAGAUU
A-108589.2
AAUCUUUGUUUUCCGGGAUUGCA
1164-1186
AD-52779.1
A-109013.1
CUACUUGGGAUCACAAAGCAA
A-108605.2
UUGCUUUGUGAUCCCAAGUAGAA
1194-1216
AD-52780.1
A-109021.1
ACAACCUAAAUGGUAAAUAUA
A-108621.2
UAUAUUUACCAUUUAGGUUGUUU
1281-1303
AD-52781.1
A-109029.1
AUCCAUCCAACAGAUUCAGAA
A-108637.2
UUCUGAAUCUGUUGGAUGGAUCA
1400-1422
AD-52782.1
A-109037.1
AACUGAGGCAAAUUUAAAAGA
A-108653.2
UCUUUUAAAUUUGCCUCAGUUCA
1432-
1454_G21A
AD-52783.1
A-109045.1
AGAGUAUGUGUAAAAAUCUGU
A-108669.2
ACAGAUUUUUACACAUACUCUGU
1913-1935
AD-52784.1
A-109006.1
AAUCCCGGAAAACAAAGAUUU
A-108591.2
AAAUCUUUGUUUUCCGGGAUUGC
1165-1187
AD-52785.1
A-109014.1
UACUUGGGAUCACAAAGCAAA
A-108607.2
UUUGCUUUGUGAUCCCAAGUAGA
1195-1217
AD-52786.1
A-109022.1
CAACCUAAAUGGUAAAUAUAA
A-108623.2
UUAUAUUUACCAUUUAGGUUGUU
1282-1304
AD-52787.1
A-109030.1
UUGAAUGAACUGAGGCAAAUU
A-108639.2
AAUUUGCCUCAGUUCAUUCAAAG
1425-1447
AD-52788.1
A-109038.1
ACUGAGGCAAAUUUAAAAGGA
A-108655.2
UCCUUUUAAAUUUGCCUCAGUUC
1433-
1455_C21A
AD-52789.1
A-109046.1
GAGUAUGUGUAAAAAUCUGUA
A-108671.2
UACAGAUUUUUACACAUACUCUG
1914-1936
AD-52791.1
A-109015.1
ACUUGGGAUCACAAAGCAAAA
A-108609.2
UUUUGCUUUGUGAUCCCAAGUAG
1196-1218
AD-52792.1
A-109023.1
AUGGUAAAUAUAACAAACCAA
A-108625.2
UUGGUUUGUUAUAUUUACCAUUU
1290-1312
AD-52793.1
A-109031.1
UGAAUGAACUGAGGCAAAUUU
A-108641.2
AAAUUUGCCUCAGUUCAUUCAAA
1426-1448
AD-52794.1
A-109039.1
CUGAGGCAAAUUUAAAAGGCA
A-108657.2
UGCCUUUUAAAUUUGCCUCAGUU
1434-1456
AD-52795.1
A-109047.1
AGUAUGUGUAAAAAUCUGUAA
A-108673.2
UUACAGAUUUUUACACAUACUCU
1915-1937
AD-52796.1
A-109008.1
GAAAACAAAGAUUUGGUGUUU
A-108595.2
AAACACCAAAUCUUUGUUUUCCG
1172-1194
AD-52797.1
A-109016.1
AGUGUGGAGAAAACAACCUAA
A-108611.2
UUAGGUUGUUUUCUCCACACUCA
1269-1291
AD-52798.1
A-109024.1
GUCUCAAAAUGGAAGGUUAUA
A-108627.2
UAUAACCUUCCAUUUUGAGACUU
1354-1376
AD-52799.1
A-109032.1
GAAUGAACUGAGGCAAAUUUA
A-108643.2
UAAAUUUGCCUCAGUUCAUUCAA
1427-1449
AD-52800.1
A-109040.1
UGAGGCAAAUUUAAAAGGCAA
A-108659.2
UUGCCUUUUAAAUUUGCCUCAGU
1435-1457
AD-52801.1
A-109048.1
GUAUGUGUAAAAAUCUGUAAU
A-108675.2
AUUACAGAUUUUUACACAUACUC
1916-1938
AD-52802.1
A-109009.1
AAAACAAAGAUUUGGUGUUUU
A-108597.2
AAAACACCAAAUCUUUGUUUUCC
1173-1195
AD-52803.1
A-109017.1
GUGUGGAGAAAACAACCUAAA
A-108613.2
UUUAGGUUGUUUUCUCCACACUC
1270-1292
AD-52804.1
A-109025.1
AUGGAAGGUUAUACUCUAUAA
A-108629.2
UUAUAGAGUAUAACCUUCCAUUU
1362-1384
AD-52805.1
A-109033.1
AAUGAACUGAGGCAAAUUUAA
A-108645.2
UUAAAUUUGCCUCAGUUCAUUCA
1428-1450
AD-52806.1
A-109041.1
GAGGCAAAUUUAAAAGGCAAU
A-108661.2
AUUGCCUUUUAAAUUUGCCUCAG
1436-1458
AD-52807.1
A-109049.1
UAUGUGUAAAAAUCUGUAAUA
A-108677.2
UAUUACAGAUUUUUACACAUACU
1917-1939
AD-52808.1
A-109010.1
ACAAAGAUUUGGUGUUUUCUA
A-108599.2
UAGAAAACACCAAAUCUUUGUUU
1176-1198
AD-52809.1
A-109018.1
UGUGGAGAAAACAACCUAAAU
A-108615.2
AUUUAGGUUGUUUUCUCCACACU
1271-1293
AD-52810.1
A-109026.1
UGGAAGGUUAUACUCUAUAAA
A-108631.2
UUUAUAGAGUAUAACCUUCCAUU
1363-1385
AD-52811.1
A-109034.1
AUGAACUGAGGCAAAUUUAAA
A-108647.2
UUUAAAUUUGCCUCAGUUCAUUC
1429-1451
AD-52812.1
A-109042.1
AGGCAAAUUUAAAAGGCAAUA
A-108663.2
UAUUGCCUUUUAAAUUUGCCUCA
1437-1459
AD-52813.1
A-109011.1
AAGAUUUGGUGUUUUCUACUU
A-108601.2
AAGUAGAAAACACCAAAUCUUUG
1179-1201
AD-52814.1
A-109019.1
AAACAACCUAAAUGGUAAAUA
A-108617.2
UAUUUACCAUUUAGGUUGUUUUC
1279-1301
AD-52815.1
A-109027.1
AUACUCUAUAAAAUCAACCAA
A-108633.2
UUGGUUGAUUUUAUAGAGUAUAA
1372-1394
AD-52816.1
A-109035.1
UGAACUGAGGCAAAUUUAAAA
A-108649.2
UUUUAAAUUUGCCUCAGUUCAUU
1430-1452
AD-52817.1
A-109043.1
GGCAAAUUUAAAAGGCAAUAA
A-108665.2
UUAUUGCCUUUUAAAUUUGCCUC
1438-1460
AD-52818.1
A-109012.1
UUUUCUACUUGGGAUCACAAA
A-108603.2
UUUGUGAUCCCAAGUAGAAAACA
1190-1212
AD-52819.1
A-109020.1
AACAACCUAAAUGGUAAAUAU
A-108619.2
AUAUUUACCAUUUAGGUUGUUUU
1280-1302
AD-52820.1
A-109028.1
UACUCUAUAAAAUCAACCAAA
A-108635.2
UUUGGUUGAUUUUAUAGAGUAUA
1373-1395
AD-52821.1
A-109036.1
GAACUGAGGCAAAUUUAAAAA
A-108651.2
UUUUUAAAUUUGCCUCAGUUCAU
1431-
1453_G21A
AD-52822.1
A-109044.1
CAGAGUAUGUGUAAAAAUCUU
A-108667.2
AAGAUUUUUACACAUACUCUGUG
1912-1934
_G21U
TABLE 10
Modified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences are the same as the sequences
listed in Table 8 except that they do not
contain GalNal conjugation.
Se-
Se-
quence
quence
(SEQ
(SEQ
ID
ID
NOS
NOS
1366-
1547-
1546,
1727,
re-
re-
spec-
spec-
tive-
tive-
ly,
ly,
in
in
order
order
of
Anti-
of
Sense
ap-
sense
ap-
Duplex
Oligo
pear-
Oligo
pear-
Name
Name
ance)
Name
ance)
AD-52637.1
A-108817.1
UfcAf
A-108307.2
aAfaG
cAfaU
faAfg
fuAfA
GfaGf
fGfcU
cuuAf
fcCfu
aUfuG
UfcUf
fuGfa
uUf
sAfsc
AD-52638.1
A-108825.1
UfuAf
A-108323.2
aAfaU
uUfgU
faAfc
fuCfC
UfaGf
fUfcU
aggAf
faGfu
aCfaA
UfaUf
fuAfa
uUf
sAfsa
AD-52639.1
A-108833.1
GfcUf
A-108339.2
uUfuU
aUfgU
faCfa
fuAfG
UfcGf
fAfcG
ucuAf
faUfg
aCfaU
UfaAf
faGfc
aAf
sAfsa
AD-52640.1
A-108841.1
GfgAf
A-108355.2
aAfaG
cAfuG
fuCfu
fgUfC
UfuAf
fUfuA
agaCf
faAfg
cAfuG
AfcUf
fuCfc
uUf
sCfsa
AD-52641.1
A-108849.1
CfaAf
A-108371.2
aUfcA
aAfaC
faAfu
fuCfA
AfuGf
fAfcA
uugAf
fuAfu
gUfuU
UfuGf
fuUfg
aUf
sAfsa
AD-52642.1
A-108857.1
AfcCf
A-108387.2
uUfcU
aGfuG
fuCfu
faAfA
UfuGf
fUfcA
auuUf
faAfg
cAfcU
AfaGf
fgGfu
aAf
sUfsu
AD-52643.1
A-108818.1
CfaCf
A-108309.2
aAfaA
aAfuU
fgAfa
faAfG
GfgAf
fCfuC
gcuUf
fcUfu
aAfuU
CfuUf
fgUfg
uUf
sAfsa
AD-52645.1
A-108834.1
CfuAf
A-108341.2
uUfuU
uGfuU
fuAfc
faGfA
AfuCf
fCfgA
gucUf
fuGfu
aAfcA
AfaAf
fuAfg
aAf
sCfsa
AD-52647.1
A-108850.1
UfcAf
A-108373.2
aAfgA
aCfaU
fcUfg
faUfU
AfuCf
fUfgA
aaaUf
fuCfa
aUfgU
GfuCf
fuGfa
uUf
sGfsu
AD-52648.1
A-108858.1
AfaCf
A-108389.2
uAfuA
uGfaG
fuGfu
faAfG
AfgUf
fAfaC
ucuUf
fuAfc
cUfcA
AfuAf
fgUfu
uAf
sCfsc
AD-52649.1
A-108819.1
AfcAf
A-108311.2
aAfaA
aUfuA
faGfa
faGfC
AfgGf
fUfcC
agcUf
fuUfc
uAfaU
UfuUf
fuGfu
uUf
sGfsa
AD-52650.1
A-108827.1
CfuCf
A-108327.2
aUfcU
cAfgA
fuGfa
fgCfC
UfuUf
fAfaA
uggCf
faUfc
uCfuG
AfaGf
fgAfg
aUf
sAfsu
AD-52651.1
A-108835.1
CfgAf
A-108343.2
uUfgG
uGfuA
fcUfa
faAfA
AfaAf
fAfuU
uuuUf
fuUfa
uAfcA
GfcCf
fuCfg
aAf
sUfsc
AD-52652.1
A-108843.1
GfuCf
A-108359.2
uAfuG
uUfaA
fgAfc
faGfA
AfaAf
fCfuU
gucUf
fuGfu
uUfaA
CfcAf
fgAfc
uAf
sCfsa
AD-52653.1
A-108851.1
CfaAf
A-108375.2
aAfaG
cAfuA
faCfu
fuUfU
GfaUf
fGfaU
caaAf
fcAfg
uAfuG
UfcUf
fuUfg
uUf
sAfsg
AD-52654.1
A-108859.1
AfcUf
A-108391.2
uUfaU
gAfgA
faUfg
faGfA
UfaGf
fAfcU
uucUf
faCfa
uCfuC
UfaUf
faGfu
aAf
sUfsc
AD-52656.1
A-108828.1
CfcAf
A-108329.2
aAfaU
gAfgC
fcUfu
fcAfA
GfaUf
fAfaU
uuuGf
fcAfa
gCfuC
GfaUf
fuGfg
uUf
sAfsg
AD-52657.1
A-108836.1
GfaUf
A-108345.2
aUfuG
gUfaA
fgCfu
faAfA
AfaAf
fUfuU
auuUf
fuAfg
uUfaC
CfcAf
faUfc
aUf
sGfsu
AD-52658.1
A-108844.1
UfcUf
A-108361.2
uUfaU
uAfaA
fgGfa
fgAfC
CfaAf
fUfuU
aguCf
fgUfc
uUfuA
CfaUf
faGfa
aAf
sCfsc
AD-52659.1
A-108852.1
AfaCf
A-108377.2
aAfaA
aUfaU
fgAfc
fuUfG
UfgAf
fAfuC
ucaAf
faGfu
aUfaU
CfuUf
fgUfu
uUf
sGfsa
AD-52660.1
A-108860.1
CfuGf
A-108393.2
uUfuA
aGfaA
fuAfu
fgAfA
GfuAf
fCfuA
guuCf
fcAfu
uUfcU
AfuAf
fcAfg
aAf
sUfsu
AD-52661.1
A-108821.1
AfaUf
A-108315.2
aUfaA
uAfaG
faAfa
fcUfC
GfaAf
fCfuU
ggaGf
fcUfu
cUfuA
UfuUf
faUfu
aUf
sGfsu
AD-52662.1
A-108829.1
AfaAf
A-108331.2
uAfaC
uCfaA
faUfa
fgAfU
GfcAf
fUfuG
aauCf
fcUfa
uUfgA
UfgUf
fuUfu
uAf
sUfsg
AD-52663.1
A-108837.1
UfuCf
A-108347.2
uAfaG
aGfuU
faCfc
fgGfG
AfuGf
fAfcA
uccCf
fuGfg
aAfcU
UfcUf
fgAfa
uAf
sGfsg
AD-52664.1
A-108845.1
GfgGf
A-108363.2
aAfuA
cCfaA
fuGfu
faUfU
CfaUf
fAfaU
uaaUf
fgAfc
uUfgG
AfuAf
fcCfc
uUf
sUfsu
AD-52665.1
A-108853.1
AfcAf
A-108379.2
aAfaA
uAfuU
faGfa
fuGfA
CfuGf
fUfcA
aucAf
fgUfc
aAfuA
UfuUf
fuGfu
uUf
sUfsg
AD-52666.1
A-108861.1
AfgAf
A-108395.2
uUfgU
aCfuA
faGfu
fcAfU
UfuAf
fAfuA
uauGf
faAfc
uAfgU
UfaCf
fuCfu
aAf
sUfsc
AD-52667.1
A-108822.1
AfuUf
A-108317.2
aAfuA
aAfgC
faAfa
fuCfC
AfgAf
fUfuC
aggAf
fuUfu
gCfuU
UfuAf
faAfu
uUf
sUfsg
AD-52668.1
A-108830.1
AfgAf
A-108333.2
aUfcG
uUfuG
fuCfu
fcUfA
AfaCf
fUfgU
auaGf
fuAfg
cAfaA
AfcGf
fuCfu
aUf
sUfsg
AD-52669.1
A-108838.1
UfcAf
A-108349.2
uUfaA
gUfuG
fgAfc
fgGfA
CfaUf
fCfaU
gucCf
fgGfu
cAfaC
CfuUf
fuGfa
aAf
sAfsg
AD-52670.1
A-108846.1
GfgCf
A-108365.2
aAfaU
cAfaA
faUfg
fuUfA
UfcAf
fAfuG
uuaAf
faCfa
uUfuG
UfaUf
fgCfc
uUf
sCfsu
AD-52671.1
A-108854.1
CfaUf
A-108381.2
uAfaA
aUfuU
faAfg
fgAfU
AfcUf
fCfaG
gauCf
fuCfu
aAfaU
UfuUf
faUfg
uAf
sUfsu
AD-52672.1
A-108862.1
UfaCf
A-108397.2
uUfgA
aUfaU
fcUfu
faAfA
GfuAf
fCfuA
guuUf
fcAfa
aUfaU
GfuCf
fgUfa
aAf
sGfsu
AD-52673.1
A-108823.1
UfuUf
A-108319.2
aUfaA
uAfuU
fcUfa
fgUfU
GfaGf
fCfcU
gaaCf
fcUfa
aAfuA
GfuUf
faAfa
aUf
sAfsg
AD-52674.1
A-108831.1
UfuGf
A-108335.2
uUfaC
cUfaU
faUfc
fgUfU
GfuCf
fAfgA
uaaCf
fcGfa
aUfaG
UfgUf
fcAfa
aAf
sAfsu
AD-52675.1
A-108839.1
CfaGf
A-108351.2
uUfuA
uUfgG
faGfa
fgAfC
CfcAf
fAfuG
uguCf
fgUfc
cCfaA
UfuAf
fcUfg
aAf
sAfsa
AD-52676.1
A-108847.1
AfaAf
A-108367.2
uUfuG
uUfaA
faAfa
fuGfA
UfaUf
fCfaU
gucAf
faUfu
uUfaA
UfcAf
fuUfu
aAf
sGfsg
AD-52677.1
A-108855.1
GfaUf
A-108383.2
uAfgA
cAfgU
fuCfa
fcUfU
UfaAf
fUfuU
aaaGf
faUfg
aCfuG
AfuCf
faUfc
uAf
sAfsa
AD-52678.1
A-108863.1
AfcAf
A-108399.2
uUfuG
uAfuA
faCfu
faAfC
UfgUf
fUfaC
aguUf
faAfg
uAfuA
UfcAf
fuGfu
aAf
sAfsg
AD-52679.1
A-108824.1
UfuUf
A-108321.2
aAfuA
aUfuG
faCfu
fuUfC
AfgAf
fCfuC
ggaAf
fuAfg
cAfaU
UfuAf
faAfa
uUf
sAfsa
AD-52680.1
A-108832.1
UfgCf
A-108337.2
uUfuA
uAfuG
fcAfu
fuUfA
CfgUf
fGfaC
cuaAf
fgAfu
cAfuA
GfuAf
fgCfa
aAf
sAfsa
AD-52681.1
A-108840.1
GfgGf
A-108353.2
aAfgU
aCfaU
fcUfu
fgGfU
UfaAf
fCfuU
gacCf
faAfa
aUfgU
GfaCf
fcCfc
uUf
sAfsa
AD-52682.1
A-108848.1
UfgAf
A-108369.2
uUfgA
cAfuA
fgUfu
fuUfU
UfuUf
fCfaA
gaaAf
faAfa
uAfuG
CfuCf
fuCfa
aAf
sUfsu
AD-52683.1
A-108856.1
AfuCf
A-108385.2
aUfaG
aGfuC
faUfc
fuUfU
AfuAf
fUfuA
aaaAf
fuGfa
gAfcU
UfcUf
fgAfu
aUf
sCfsa
AD-52684.1
A-108864.1
CfaUf
A-108401.2
uUfuU
aUfaA
fgAfc
faCfU
UfuGf
fAfcA
uagUf
faGfu
uUfaU
CfaAf
faUfg
aAf
sUfsa
AD-52685.1
A-108872.1
CfuUf
A-108417.2
aAfgU
gAfaC
fuUfu
fuCfA
GfaGf
fAfcU
uugAf
fcAfa
gUfuC
AfaCf
faAfg
uUf
sUfsg
AD-52686.1
A-108880.1
CfuAf
A-108433.2
uUfuC
cUfuC
faCfu
faAfC
UfuUf
fAfaA
uguUf
faAfg
gAfaG
UfgAf
fuAfg
aAf
sAfsa
AD-52687.1
A-108888.1
AfaGf
A-108449.2
uUfaA
aGfcA
fgUfu
faCfU
AfgUf
fAfaC
uagUf
fuAfa
uGfcU
CfuUf
fcUfu
aAf
sCfsu
AD-52688.1
A-108896.1
AfaAf
A-108465.2
uUfuG
cAfaG
faUfg
faUfA
CfuAf
fAfuA
uuaUf
fgCfa
cUfuG
UfcAf
fuUfu
aAf
sUfsu
AD-52689.1
A-108904.1
GfcAf
A-108481.2
aUfuU
uAfgU
fcUfu
fcAfA
UfuAf
fAfuA
uuuGf
faAfa
aCfuA
GfaAf
fuGfc
aUf
sUfsg
AD-52690.1
A-108865.1
AfuAf
A-108403.2
uUfuU
uAfaA
fuGfa
fcUfA
CfuUf
fCfaA
guaGf
fgUfc
uUfuA
AfaAf
fuAfu
aAf
sGfsu
AD-52691.1
A-108873.1
GfaAf
A-108419.2
uUfcA
cUfcA
faGfu
faCfU
UfuUf
fCfaA
gagUf
faAfc
uGfaG
UfuGf
fuUfc
aAf
sAfsa
AD-52692.1
A-108881.1
UfaCf
A-108435.2
aUfuU
uUfcA
fcAfc
faCfA
UfuUf
fAfaA
uugUf
faGfu
uGfaA
GfaAf
fgUfa
aUf
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fcAfu
gAfcU
UfaAf
faCfc
uUf
sUfsg
AD-52774.1
A-108973.1
AfuCf
A-108525.2
uUfuG
gAfaU
fuGfa
faGfA
UfcCf
fUfgG
aucUf
faUfc
aUfuC
AfcAf
fgAfu
aAf
sGfsu
AD-52775.1
A-108981.1
CfcUf
A-108541.2
aUfgG
aGfaG
faGfu
faAfG
AfuAf
fAfuA
ucuUf
fuAfc
cUfcU
UfcCf
faGfg
aUf
sCfsc
AD-52776.1
A-108989.1
GfuUf
A-108557.2
uUfgU
gGfaA
fcUfu
fgAfC
UfcCf
fUfgG
aguCf
faAfa
uUfcC
GfaCf
faAfc
aAf
sUfsc
AD-52777.1
A-108997.1
AfcAf
A-108573.2
aAfaG
uUfaU
faAfu
faUfU
AfuUf
fGfaA
caaUf
fuAfu
aUfaA
UfcUf
fuGfu
uUf
sUfsu
AD-52778.1
A-109005.1
CfaAf
A-108589.2
aAfuC
uCfcC
fuUfu
fgGfA
GfuUf
fAfaA
uucCf
fcAfa
gGfgA
AfgAf
fuUfg
uUf
sCfsa
AD-52779.1
A-109013.1
CfuAf
A-108605.2
uUfgC
cUfuG
fuUfu
fgGfA
GfuGf
fUfcA
aucCf
fcAfa
cAfaG
AfgCf
fuAfg
aAf
sAfsa
AD-52780.1
A-109021.1
AfcAf
A-108621.2
uAfuA
aCfcU
fuUfu
faAfA
AfcCf
fUfgG
auuUf
fuAfa
aGfgU
AfuAf
fuGfu
uAf
sUfsu
AD-52781.1
A-109029.1
AfuCf
A-108637.2
uUfcU
cAfuC
fgAfa
fcAfA
UfcUf
fCfaG
guuGf
faUfu
gAfuG
CfaGf
fgAfu
aAf
sCfsa
AD-52782.1
A-109037.1
AfaCf
A-108653.2
uCfuU
uGfaG
fuUfa
fgCfA
AfaUf
fAfaU
uugCf
fuUfa
cUfcA
AfaAf
fgUfu
gAf
sCfsa
AD-52783.1
A-109045.1
AfgAf
A-108669.2
aCfaG
gUfaU
faUfu
fgUfG
UfuUf
fUfaA
acaCf
faAfa
aUfaC
UfcUf
fuCfu
gUf
sGfsu
AD-52784.1
A-109006.1
AfaUf
A-108591.2
aAfaU
cCfcG
fcUfu
fgAfA
UfgUf
fAfaC
uuuCf
faAfa
cGfgG
GfaUf
faUfu
uUf
sGfsc
AD-52785.1
A-109014.1
UfaCf
A-108607.2
uUfuG
uUfgG
fcUfu
fgAfU
UfgUf
fCfaC
gauCf
faAfa
cCfaA
GfcAf
fgUfa
aAf
sGfsa
AD-52786.1
A-109022.1
CfaAf
A-108623.2
uUfaU
cCfuA
faUfu
faAfU
UfaCf
fGfgU
cauUf
faAfa
uAfgG
UfaUf
fuUfg
aAf
sUfsu
AD-52787.1
A-109030.1
UfuGf
A-108639.2
aAfuU
aAfuG
fuGfc
faAfC
CfuCf
fUfgA
aguUf
fgGfc
cAfuU
AfaAf
fcAfa
uUf
sAfsg
AD-52788.1
A-109038.1
AfcUf
A-108655.2
uCfcU
gAfgG
fuUfu
fcAfA
AfaAf
fAfuU
uuuGf
fuAfa
cCfuC
AfaGf
faGfu
gAf
sUfsc
AD-52789.1
A-109046.1
GfaGf
A-108671.2
uAfcA
uAfuG
fgAfu
fuGfU
UfuUf
fAfaA
uacAf
faAfu
cAfuA
CfuGf
fcUfc
uAf
sUfsg
AD-52791.1
A-109015.1
AfcUf
A-108609.2
uUfuU
uGfgG
fgCfu
faUfC
UfuGf
fAfcA
ugaUf
faAfg
cCfcA
CfaAf
faGfu
aAf
sAfsg
AD-52792.1
A-109023.1
AfuGf
A-108625.2
uUfgG
gUfaA
fuUfu
faUfA
GfuUf
fUfaA
auaUf
fcAfa
uUfaC
AfcCf
fcAfu
aAf
sUfsu
AD-52793.1
A-109031.1
UfgAf
A-108641.2
aAfaU
aUfgA
fuUfg
faCfU
CfcUf
fGfaG
cagUf
fgCfa
uCfaU
AfaUf
fuCfa
uUf
sAfsa
AD-52794.1
A-109039.1
CfuGf
A-108657.2
uGfcC
aGfgC
fuUfu
faAfA
UfaAf
fUfuU
auuUf
faAfa
gCfcU
AfgGf
fcAfg
cAf
sUfsu
AD-52795.1
A-109047.1
AfgUf
A-108673.2
uUfaC
aUfgU
faGfa
fgUfA
UfuUf
fAfaA
uuaCf
faUfc
aCfaU
UfgUf
faCfu
aAf
sCfsu
AD-52796.1
A-109008.1
GfaAf
A-108595.2
aAfaC
aAfcA
faCfc
faAfG
AfaAf
fAfuU
ucuUf
fuGfg
uGfuU
UfgUf
fuUfc
uUf
sCfsg
AD-52797.1
A-109016.1
AfgUf
A-108611.2
uUfaG
gUfgG
fgUfu
faGfA
GfuUf
fAfaA
uucUf
fcAfa
cCfaC
CfcUf
faCfu
aAf
sCfsa
AD-52798.1
A-109024.1
GfuCf
A-108627.2
uAfuA
uCfaA
faCfc
faAfU
UfuCf
fGfgA
cauUf
faGfg
uUfgA
UfuAf
fgAfc
uAf
sUfsu
AD-52799.1
A-109032.1
GfaAf
A-108643.2
uAfaA
uGfaA
fuUfu
fcUfG
GfcCf
fAfgG
ucaGf
fcAfa
uUfcA
AfuUf
fuUfc
uAf
sAfsa
AD-52800.1
A-109040.1
UfgAf
A-108659.2
uUfgC
gGfcA
fcUfu
faAfU
UfuAf
fUfuA
aauUf
faAfa
uGfcC
GfgCf
fuCfa
aAf
sGfsu
AD-52801.1
A-109048.1
GfuAf
A-108675.2
aUfuA
uGfuG
fcAfg
fuAfA
AfuUf
fAfaA
uuuAf
fuCfu
cAfcA
GfuAf
fuAfc
aUf
sUfsc
AD-52802.1
A-109009.1
AfaAf
A-108597.2
aAfaA
aCfaA
fcAfc
faGfA
CfaAf
fUfuU
aucUf
fgGfu
uUfgU
GfuUf
fuUfu
uUf
sCfsc
AD-52803.1
A-109017.1
GfuGf
A-108613.2
uUfuA
uGfgA
fgGfu
fgAfA
UfgUf
fAfaC
uuuCf
faAfc
uCfcA
CfuAf
fcAfc
aAf
sUfsc
AD-52804.1
A-109025.1
AfuGf
A-108629.2
uUfaU
gAfaG
faGfa
fgUfU
GfuAf
fAfuA
uaaCf
fcUfc
cUfuC
UfaUf
fcAfu
aAf
sUfsu
AD-52805.1
A-109033.1
AfaUf
A-108645.2
uUfaA
gAfaC
faUfu
fuGfA
UfgCf
fGfgC
cucAf
faAfa
gUfuC
UfuUf
faUfu
aAf
sCfsa
AD-52806.1
A-109041.1
GfaGf
A-108661.2
aUfuG
gCfaA
fcCfu
faUfU
UfuUf
fUfaA
aaaUf
faAfg
uUfgC
GfcAf
fcUfc
aUf
sAfsg
AD-52807.1
A-109049.1
UfaUf
A-108677.2
uAfuU
gUfgU
faCfa
faAfA
GfaUf
fAfaU
uuuUf
fcUfg
aCfaC
UfaAf
faUfa
uAf
sCfsu
AD-52808.1
A-109010.1
AfcAf
A-108599.2
uAfgA
aAfgA
faAfa
fuUfU
CfaCf
fGfgU
caaAf
fgUfu
uCfuU
UfuCf
fuGfu
uAf
sUfsu
AD-52809.1
A-109018.1
UfgUf
A-108615.2
aUfuU
gGfaG
faGfg
faAfA
UfuGf
fAfcA
uuuUf
faCfc
cUfcC
UfaAf
faCfa
aUf
sCfsu
AD-52810.1
A-109026.1
UfgGf
A-108631.2
uUfuA
aAfgG
fuAfg
fuUfA
AfgUf
fUfaC
auaAf
fuCfu
cCfuU
AfuAf
fcCfa
aAf
sUfsu
AD-52811.1
A-109034.1
AfuGf
A-108647.2
uUfuA
aAfcU
faAfu
fgAfG
UfuGf
fGfcA
ccuCf
faAfu
aGfuU
UfuAf
fcAfu
aAf
sUfsc
AD-52812.1
A-109042.1
AfgGf
A-108663.2
uAfuU
cAfaA
fgCfc
fuUfU
UfuUf
fAfaA
uaaAf
faGfg
uUfuG
CfaAf
fcCfu
uAf
sCfsa
AD-52813.1
A-109011.1
AfaGf
A-108601.2
aAfgU
aUfuU
faGfa
fgGfU
AfaAf
fGfuU
cacCf
fuUfc
aAfaU
UfaCf
fcUfu
uUf
sUfsg
AD-52814.1
A-109019.1
AfaAf
A-108617.2
uAfuU
cAfaC
fuAfc
fcUfA
CfaUf
fAfaU
uuaGf
fgGfu
gUfuG
AfaAf
fuUfu
uAf
sUfsc
AD-52815.1
A-109027.1
AfuAf
A-108633.2
uUfgG
cUfcU
fuUfg
faUfA
AfuUf
fAfaA
uuaUf
fuCfa
aGfaG
AfcCf
fuAfu
aAf
sAfsa
AD-52816.1
A-109035.1
UfgAf
A-108649.2
uUfuU
aCfuG
faAfa
faGfG
UfuUf
fCfaA
gccUf
faUfu
cAfgU
UfaAf
fuCfa
aAf
sUfsu
AD-52817.1
A-109043.1
GfgCf
A-108665.2
uUfaU
aAfaU
fuGfc
fuUfA
CfuUf
fAfaA
uuaAf
fgGfc
aUfuU
AfaUf
fgCfc
aAf
sUfsc
AD-52818.1
A-109012.1
UfuUf
A-108603.2
uUfuG
uCfuA
fuGfa
fcUfU
UfcCf
fGfgG
caaGf
faUfc
uAfgA
AfcAf
faAfa
aAf
sCfsa
AD-52819.1
A-109020.1
AfaCf
A-108619.2
aUfaU
aAfcC
fuUfa
fuAfA
CfcAf
fAfuG
uuuAf
fgUfa
gGfuU
AfaUf
fgUfu
aUf
sUfsu
AD-52820.1
A-109028.1
UfaCf
A-108635.2
uUfuG
uCfuA
fgUfu
fuAfA
GfaUf
fAfaU
uuuAf
fcAfa
uAfgA
CfcAf
fgUfa
aAf
sUfsa
AD-52821.1
A-109036.1
GfaAf
A-108651.2
uUfuU
cUfgA
fuAfa
fgGfC
AfuUf
fAfaA
ugcCf
fuUfu
uCfaG
AfaAf
fuUfc
aAf
sAfsu
AD-52822.1
A-109044.1
CfaGf
A-108667.2
aAfgA
aGfuA
fuUfu
fuGfU
UfuAf
fGfuA
cacAf
faAfa
uAfcU
AfuCf
fcUfg
uUf
sUfsg
TABLE 11
Results of single dose screen using ANGPTL3 GalNac-conjugated dsRNA
Modified siRNAs were tested by transfection in Hep3b cells and by free-uptake in primary
cynomolgus monkey (PCH) cells at the above-stated doses.
500 nM
100 nM
10 nM
PCH
PCH
PCH
STDEV
STDEV
STDEV
STDEV
STDEV
10 nM
0.1 nM
Celsis
Celsis
Celsis
10 nM
0.1 nM
500 nM
100 nM
10 nM
DUPLEX ID
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
AD1955/naïve FU
0.93
0.93
1.01
0.91
1.17
0.02
0.08
0.09
0.00
0.07
AD1955/naïve FU
1.02
1.09
1.07
1.07
0.92
0.06
0.04
0.02
0.00
0.03
AD1955/naïve FU
1.06
0.99
0.93
1.02
0.93
0.03
0.00
0.09
0.01
0.02
AD1955/naïve FU
1.05
0.90
1.05
1.03
1.03
0.04
0.02
0.01
0.05
0.01
AD1955/naïve FU
1.06
1.08
0.90
0.97
1.03
0.02
0.01
0.02
0.04
0.09
AD1955/naïve FU
0.90
1.03
1.05
1.00
0.94
0.04
0.03
0.01
0.04
0.05
AD-45165 (TTR)
0.91
0.98
1.06
0.98
0.96
0.05
0.01
0.05
0.00
0.00
AD-52953.1
0.06
0.34
0.15
0.17
0.46
0.00
0.01
0.00
0.01
0.01
AD-52954.1
0.09
0.39
0.17
0.20
0.55
0.00
0.01
0.00
0.01
0.00
AD-52955.1
0.11
0.59
0.38
0.41
0.75
0.01
0.04
0.02
0.01
0.12
AD-52956.1
0.31
0.94
0.79
0.94
1.17
0.01
0.00
0.02
0.06
0.02
AD-52957.1
0.13
0.61
0.35
0.38
0.73
0.01
0.00
0.01
0.00
0.04
AD-52958.1
0.19
0.74
0.66
0.71
0.97
0.01
0.01
0.02
0.07
0.06
AD-52960.1
0.14
0.59
0.31
0.32
0.55
0.01
0.01
0.00
0.02
0.02
AD-52961.1
0.05
0.66
0.27
0.24
0.49
0.00
0.00
0.00
0.02
0.02
AD-52962.1
0.83
0.89
1.03
1.02
1.26
0.02
0.05
0.07
0.07
0.07
AD-52963.1
0.07
0.72
0.46
0.56
0.91
0.00
0.00
0.00
0.00
0.06
AD-52964.1
0.13
0.73
0.41
0.47
0.68
0.01
0.03
0.02
0.03
0.01
AD-52965.1
0.07
0.44
0.16
0.18
0.43
0.00
0.01
0.00
0.01
0.01
AD-52966.1
0.12
0.76
0.67
0.72
0.96
0.00
0.02
0.05
0.01
0.01
AD-52967.1
0.10
0.75
0.44
0.58
0.89
0.01
0.04
0.02
0.03
0.04
AD-52968.1
1.01
0.96
0.87
0.91
1.15
0.00
0.01
0.09
0.03
0.02
AD-52969.1
0.04
0.46
0.22
0.29
0.59
0.00
0.00
0.01
0.02
0.04
AD-52970.1
0.06
0.45
0.27
0.30
0.51
0.00
0.00
0.01
0.02
0.00
AD-52971.1
0.08
0.55
0.20
0.22
0.45
0.00
0.00
0.01
0.02
0.05
AD-52972.1
0.10
0.73
0.41
0.49
0.81
0.00
0.01
0.01
0.02
0.01
AD-52973.1
0.11
0.73
0.36
0.46
0.75
0.01
0.01
0.03
0.02
0.02
AD-52974.1
1.00
0.95
1.00
1.09
1.27
0.01
0.01
0.08
0.05
0.06
AD-52975.1
0.07
0.54
0.25
0.34
0.66
0.00
0.01
0.01
0.01
0.03
AD-52976.1
0.17
0.59
0.35
0.41
0.65
0.00
0.02
0.04
0.01
0.01
AD-52977.1
0.07
0.45
0.16
0.25
0.50
0.01
0.02
0.00
0.02
0.03
AD-52978.1
0.10
0.72
0.39
0.53
0.77
0.00
0.02
0.00
0.08
0.03
AD-52979.1
0.54
0.92
0.99
1.12
1.28
0.01
0.02
0.02
0.04
0.05
AD-52980.1
0.29
0.85
0.67
0.85
1.03
0.01
0.01
0.05
0.05
0.04
AD-52981.1
0.07
0.44
0.20
0.26
0.59
0.01
0.02
0.00
0.00
0.03
AD-52982.1
0.28
0.87
0.67
0.99
1.14
0.01
0.01
0.04
0.00
0.01
AD-52983.1
0.06
0.40
0.14
0.40
0.46
0.00
0.00
0.01
0.05
0.02
AD-52984.1
0.29
0.87
0.66
0.74
1.09
0.01
0.02
0.01
0.00
0.00
AD-52985.1
0.72
0.87
0.89
1.18
1.22
0.03
0.00
0.05
0.03
0.16
AD-52986.1
0.08
0.47
0.24
0.30
0.48
0.00
0.02
0.02
0.00
0.06
AD-52987.1
0.16
0.83
0.42
0.73
1.09
0.00
0.00
0.01
0.02
0.02
AD-52988.1
0.11
0.73
0.42
0.60
0.96
0.01
0.04
0.00
0.00
0.10
AD-52989.1
0.05
0.48
0.15
0.42
0.46
0.00
0.02
0.00
0.02
0.00
AD-52990.1
0.14
0.86
0.33
0.45
0.77
0.00
0.01
0.00
0.02
0.05
AD-52991.1
0.16
0.86
0.58
0.69
1.05
0.00
0.00
0.02
0.00
0.02
AD-52992.1
0.08
0.65
0.42
0.56
0.90
0.00
0.01
0.02
0.01
0.00
AD-52993.1
0.13
0.87
0.53
0.76
1.08
0.02
0.03
0.04
0.04
0.00
AD-52994.1
0.10
0.52
0.28
0.33
0.53
0.01
0.00
0.02
0.00
0.01
AD-52995.1
0.06
0.56
0.19
0.41
0.60
0.00
0.01
0.04
0.02
0.05
AD-52996.1
0.09
0.68
0.26
0.47
0.68
0.00
0.03
0.01
0.04
0.01
AD-52997.1
0.59
1.03
0.87
0.51
1.25
0.05
0.01
0.00
0.01
0.01
AD-52998.1
0.09
0.79
0.44
0.55
0.85
0.00
0.00
0.04
0.03
0.10
AD-52999.1
0.08
0.57
0.17
0.36
0.84
0.01
0.00
0.01
0.02
0.00
AD-53000.1
0.38
0.94
0.58
0.67
0.85
0.01
0.02
0.03
0.03
0.02
AD-53001.1
0.05
0.48
0.21
0.18
0.40
0.00
0.00
0.01
0.00
0.05
AD-53002.1
0.07
0.65
0.43
0.48
0.80
0.00
0.05
0.04
0.01
0.02
AD-53003.1
0.05
0.46
0.31
0.34
0.56
0.01
0.01
0.00
0.02
0.05
AD-53004.1
0.05
0.36
0.29
0.66
0.57
0.00
0.01
0.03
0.35
0.02
AD-53005.1
0.05
0.72
0.32
0.58
0.83
0.01
0.00
0.01
0.29
0.00
AD-53006.1
0.21
0.82
0.66
0.77
1.03
0.01
0.00
0.02
0.07
0.02
AD-53007.1
0.12
0.76
0.55
0.73
0.74
0.01
0.00
0.00
0.08
0.20
AD-53008.1
0.07
0.68
0.28
0.36
0.84
0.00
0.02
0.01
0.05
0.03
AD-53009.1
0.10
0.61
0.48
0.60
0.91
0.00
0.02
0.01
0.01
0.06
AD-53010.1
0.05
0.58
0.47
0.54
0.84
0.00
0.02
0.00
0.02
0.03
AD-53011.1
0.07
0.65
0.29
0.34
0.84
0.00
0.03
0.07
0.01
0.04
AD-53012.1
0.06
0.55
0.36
0.45
0.70
0.00
0.03
0.02
0.02
0.00
AD-53013.1
0.11
0.85
0.59
0.70
1.01
0.00
0.00
0.03
0.03
0.02
AD-53014.1
0.16
0.78
0.61
0.78
1.11
0.00
0.02
0.01
0.05
0.00
AD-53015.1
0.03
0.35
0.25
0.37
0.46
0.01
0.01
0.01
0.00
0.01
AD-53016.1
0.03
0.56
0.40
0.58
1.01
0.00
0.01
0.02
0.06
0.09
AD-53017.1
0.07
0.71
0.64
0.78
0.98
0.00
0.01
0.01
0.05
0.00
AD-53018.1
0.30
0.96
0.75
0.97
1.14
0.00
0.02
0.02
0.03
0.05
AD-53019.1
0.27
0.99
0.77
1.05
1.31
0.00
0.01
0.01
0.04
0.00
AD-53020.1
0.04
0.64
0.32
0.45
0.69
0.00
0.00
0.03
0.02
0.03
AD-53021.1
0.04
0.68
0.36
0.48
0.70
0.01
0.01
0.02
0.07
0.00
AD-53022.1
0.05
0.76
0.36
0.59
1.04
0.01
0.01
0.02
0.03
0.06
AD-53023.1
0.10
0.83
0.69
0.84
0.97
0.01
0.01
0.06
0.02
0.01
AD-53024.1
0.09
0.44
0.23
0.23
0.44
0.00
0.00
0.03
0.01
0.02
AD-53025.1
0.09
0.87
0.58
0.80
1.09
0.00
0.03
0.01
0.04
0.04
AD-53026.1
0.05
0.60
0.35
0.46
0.77
0.01
0.01
0.02
0.05
0.03
AD-53027.1
0.02
0.32
0.26
0.30
0.45
0.00
0.01
0.02
0.03
0.02
AD-53028.1
0.19
0.82
0.77
0.95
1.04
0.01
0.04
0.05
0.01
0.03
AD-53029.1
0.02
0.52
0.32
0.41
0.72
0.00
0.00
0.01
0.02
0.07
AD-53030.1
0.09
0.42
0.15
0.16
0.46
0.00
0.00
0.00
0.00
0.02
AD-53031.1
0.12
0.79
0.63
0.73
1.04
0.02
0.05
0.02
0.04
0.03
AD-53032.1
0.12
0.71
0.41
0.59
0.90
0.01
0.00
0.02
0.04
0.00
AD-53033.1
0.02
0.48
0.20
0.21
0.51
0.00
0.02
0.02
0.01
0.00
AD-53034.1
0.04
0.52
0.31
0.36
0.71
0.00
0.01
0.07
0.02
0.01
AD-53035.1
0.02
0.63
0.34
0.50
0.85
0.00
0.02
0.03
0.00
0.03
AD-53036.1
0.10
0.57
0.31
0.35
0.65
0.01
0.01
0.03
0.03
0.01
AD-53037.1
0.08
0.47
0.27
0.36
0.60
0.00
0.02
0.01
0.03
0.01
AD-53038.1
0.05
0.85
0.48
0.63
1.08
0.00
0.05
0.00
0.02
0.05
AD-53039.1
0.08
0.82
0.45
0.64
0.97
0.00
0.01
0.01
0.03
0.00
AD-53040.1
0.05
0.79
0.46
0.62
0.97
0.01
0.01
0.01
0.05
0.06
AD-53041.1
0.06
0.72
0.59
0.61
0.86
0.00
0.01
0.05
0.06
0.03
AD-53042.1
0.08
0.85
0.30
0.35
0.81
0.01
0.00
0.00
0.03
0.03
AD-53043.1
0.63
1.00
0.92
1.04
1.07
0.03
0.00
0.06
0.03
0.07
AD-53044.1
0.05
0.91
0.35
0.61
0.97
0.01
0.01
0.01
0.04
0.02
AD-53045.1
0.20
1.00
0.85
1.00
0.98
0.00
0.03
0.04
0.01
0.04
AD-53046.1
0.07
0.70
0.44
0.62
1.12
0.00
0.01
0.03
0.00
0.09
AD-53059.1
0.35
1.04
0.75
0.85
0.86
0.01
0.01
0.03
0.02
0.04
AD-53060.1
0.34
0.85
0.72
0.96
0.82
0.00
0.01
0.02
0.01
0.02
AD-53061.1
0.17
0.94
0.36
0.37
0.59
0.00
0.00
0.02
0.00
0.02
AD-53062.1
0.09
0.76
0.43
0.47
0.69
0.01
0.01
0.01
0.03
0.01
AD-53063.1
0.06
0.48
0.18
0.16
0.25
0.00
0.01
0.01
0.01
0.02
AD-53064.1
0.07
0.59
0.22
0.22
0.48
0.01
0.02
0.01
0.02
0.06
AD-53065.1
0.08
0.97
0.45
0.39
0.64
0.01
0.01
0.02
0.01
0.01
AD-53066.1
0.12
0.99
0.73
0.67
0.88
0.01
0.03
0.01
0.01
0.05
AD-53067.1
0.12
1.08
0.59
0.60
0.79
0.00
0.12
0.01
0.01
0.03
AD-53068.1
0.09
0.98
0.46
0.59
0.83
0.00
0.03
0.04
0.07
0.05
AD-53069.1
0.04
0.69
0.35
0.43
0.59
0.00
0.01
0.01
0.04
0.01
AD-53070.1
0.17
1.12
0.88
0.83
0.98
0.00
0.01
0.04
0.00
0.01
AD-53071.1
0.07
0.70
0.23
0.23
0.43
0.00
0.00
0.02
0.00
0.01
AD-53072.1
0.10
0.90
0.49
0.48
0.75
0.01
0.05
0.00
0.01
0.02
AD-53073.1
0.07
0.63
0.27
0.30
0.43
0.00
0.00
0.01
0.01
0.00
AD-53074.1
0.07
0.88
0.46
0.49
0.62
0.01
0.08
0.01
0.06
0.03
AD-53075.1
0.05
0.76
0.29
0.35
0.50
0.01
0.01
0.00
0.02
0.03
AD-53076.1
0.09
0.80
0.31
0.40
0.54
0.01
0.01
0.02
0.05
0.02
AD-53077.1
0.07
0.96
0.29
0.28
0.49
0.00
0.03
0.00
0.01
0.01
AD-53078.1
0.16
0.95
0.51
0.51
0.70
0.00
0.04
0.01
0.01
0.06
AD-53079.1
0.08
0.96
0.59
0.67
0.83
0.00
0.02
0.01
0.03
0.01
AD-53080.1
0.04
0.63
0.20
0.22
0.43
0.00
0.01
0.00
0.01
0.01
AD-53081.1
0.16
1.02
0.63
0.75
0.87
0.00
0.09
0.00
0.02
0.05
AD-53082.1
0.06
0.94
0.50
0.52
0.66
0.01
0.06
0.02
0.03
0.03
AD-53083.1
0.14
0.87
0.48
0.50
0.80
0.01
0.02
0.04
0.06
0.01
AD-53084.1
0.12
0.95
0.50
0.47
0.72
0.01
0.03
0.04
0.00
0.00
AD-53085.1
0.27
1.02
0.68
0.81
0.99
0.01
0.01
0.01
0.05
0.02
AD-53086.1
0.05
0.60
0.26
0.25
0.48
0.00
0.01
0.03
0.00
0.01
AD-53087.1
0.05
0.56
0.32
0.39
0.53
0.00
0.01
0.01
0.03
0.02
AD-53088.1
0.09
0.89
0.53
0.69
0.87
0.00
0.01
0.02
0.04
0.02
AD-53089.1
0.29
0.97
0.58
0.57
0.78
0.01
0.00
0.02
0.02
0.02
AD-53090.1
0.13
0.86
0.56
0.55
0.73
0.00
0.01
0.01
0.03
0.00
AD-53091.1
0.12
0.82
0.27
0.35
0.66
0.00
0.03
0.03
0.01
0.07
AD-53092.1
0.05
0.66
0.26
0.29
0.42
0.00
0.01
0.02
0.04
0.02
AD-53093.1
0.08
0.68
0.36
0.44
0.55
0.00
0.02
0.03
0.04
0.10
AD-53094.1
0.32
1.00
1.05
0.92
1.11
0.02
0.01
0.01
0.00
0.03
AD-53095.1
0.14
0.77
0.29
0.29
0.49
0.00
0.02
0.00
0.01
0.01
AD-53096.1
0.30
0.96
0.61
0.57
0.73
0.03
0.01
0.02
0.02
0.01
AD-53097.1
0.37
0.97
0.67
0.82
0.86
0.01
0.01
0.01
0.02
0.01
AD-53098.1
0.06
0.65
0.22
0.30
0.43
0.00
0.03
0.03
0.00
0.01
AD-53099.1
0.34
0.99
0.61
0.81
0.91
0.00
0.00
0.04
0.02
0.06
AD-53100.1
0.31
1.04
0.95
1.03
1.00
0.02
0.01
0.06
0.02
0.17
AD-53101.1
0.46
0.93
0.63
0.69
0.78
0.00
0.01
0.04
0.03
0.04
AD-53102.1
0.23
0.80
0.60
0.55
0.66
0.00
0.03
0.01
0.02
0.03
AD-53103.1
0.05
0.61
0.27
0.32
0.50
0.01
0.02
0.00
0.01
0.00
AD-53104.1
0.13
0.80
0.64
0.68
0.77
0.00
0.02
0.03
0.01
0.05
AD-53105.1
0.15
0.77
0.43
0.65
0.77
0.01
0.03
0.02
0.02
0.05
AD-53106.1
0.16
0.87
0.72
0.70
0.83
0.01
0.02
0.00
0.00
0.04
AD-53107.1
0.19
0.95
0.62
0.65
0.90
0.00
0.02
0.01
0.03
0.04
AD-53108.1
0.22
0.94
0.60
0.68
0.81
0.00
0.01
0.00
0.03
0.04
AD-53109.1
0.16
1.01
0.82
0.78
0.96
0.01
0.08
0.04
0.01
0.07
AD-53110.1
0.10
0.86
0.79
0.77
0.94
0.00
0.05
0.03
0.01
0.05
AD-53111.1
0.22
0.78
0.94
0.85
1.04
0.01
0.01
0.01
0.01
0.07
AD-53112.1
0.09
0.96
0.64
0.65
0.86
0.01
0.02
0.07
0.07
0.00
AD-53113.1
0.10
0.97
0.71
0.77
0.88
0.01
0.05
0.01
0.02
0.01
AD-53114.1
0.19
0.83
0.48
0.52
0.66
0.01
0.01
0.02
0.01
0.00
AD-53115.1
0.10
0.59
0.42
0.44
0.66
0.01
0.03
0.04
0.00
0.02
AD-53116.1
0.11
0.87
0.82
0.85
0.95
0.00
0.05
0.05
0.05
0.05
AD-53117.1
0.52
0.64
1.21
1.00
1.08
0.01
0.03
0.09
0.04
0.07
AD-53118.1
0.19
1.04
0.60
0.72
0.94
0.00
0.07
0.02
0.05
0.06
AD-53119.1
0.06
0.77
0.44
0.47
0.64
0.01
0.03
0.00
0.01
0.01
AD-53120.1
0.10
0.97
0.78
0.89
1.01
0.01
0.04
0.05
0.01
0.04
AD-53121.1
0.23
0.80
0.58
0.69
0.90
0.01
0.02
0.04
0.02
0.06
AD-53122.1
0.09
0.80
0.90
0.94
1.09
0.01
0.07
0.02
0.04
0.10
AD-53123.1
0.27
0.74
0.95
0.93
0.97
0.00
0.01
0.03
0.01
0.08
AD-53124.1
0.08
0.81
0.33
0.34
0.61
0.01
0.02
0.00
0.01
0.01
AD-53125.1
0.08
0.82
0.34
0.38
0.58
0.00
0.02
0.00
0.01
0.07
AD-53126.1
0.15
0.95
0.70
0.86
1.06
0.01
0.04
0.05
0.02
0.00
AD-53127.1
0.21
0.81
0.62
0.75
0.91
0.02
0.04
0.01
0.03
0.00
AD-53128.1
0.08
0.79
0.80
1.14
1.09
0.00
0.06
0.04
0.01
0.03
AD-53129.1
0.48
0.78
1.05
1.00
1.10
0.00
0.01
0.06
0.01
0.03
AD-53130.1
0.25
1.08
0.63
0.72
0.88
0.01
0.02
0.00
0.01
0.00
AD-53131.1
0.14
0.96
0.54
0.57
0.81
0.02
0.02
0.05
0.01
0.04
AD-53132.1
0.03
0.54
0.24
0.27
0.49
0.00
0.02
0.02
0.00
0.01
AD-53133.1
0.12
0.76
0.50
0.67
0.93
0.00
0.03
0.01
0.01
0.06
AD-53134.1
0.28
0.86
1.14
0.81
0.97
0.01
0.04
0.05
0.02
0.04
AD-53135.1
0.47
0.74
1.03
0.94
1.09
0.01
0.03
0.04
0.07
0.04
AD-53136.1
0.09
0.99
0.64
0.69
0.94
0.01
0.05
0.01
0.05
0.02
AD-53137.1
0.08
0.75
0.39
0.39
0.59
0.01
0.03
0.00
0.00
0.00
AD-53138.1
0.04
0.71
0.33
0.34
0.60
0.00
0.02
0.00
0.03
0.00
AD-53139.1
0.11
0.76
0.55
0.66
0.84
0.01
0.01
0.06
0.01
0.02
AD-53140.1
0.09
0.71
0.64
0.71
0.86
0.00
0.04
0.01
0.02
0.02
AD-53141.1
0.24
1.09
0.77
0.91
0.93
0.00
0.01
0.00
0.06
0.00
AD-53142.1
0.13
0.95
0.55
0.70
0.82
0.01
0.03
0.03
0.04
0.02
AD-53143.1
0.13
0.91
0.67
0.83
0.94
0.01
0.00
0.03
0.03
0.07
AD-53144.1
0.10
0.72
0.54
0.69
0.84
0.01
0.03
0.01
0.03
0.00
AD-53145.1
0.08
0.72
0.70
0.78
0.88
0.01
0.03
0.01
0.08
0.02
AD-53146.1
0.83
1.07
0.85
0.96
0.98
0.01
0.06
0.00
0.05
0.00
AD-53147.1
0.08
0.56
0.27
0.34
0.47
0.00
0.01
0.01
0.01
0.01
AD-53148.1
0.06
0.81
0.61
0.68
0.74
0.01
0.00
0.03
0.06
0.05
AD-53149.1
0.23
0.86
0.71
0.83
0.92
0.01
0.02
0.06
0.02
0.03
AD-53150.1
0.41
0.70
1.03
1.09
1.03
0.03
0.06
0.03
0.04
0.01
TABLE 12
Dose response screen results for
ANGPTL3 GalNac-conjugated dsRNA sequences
A subset of active siRNAs from the single dose
screen (refer to data in Table 11) was tested in a
dose response experiment by free uptake in PCH cells.
A subset of these active siRNAs was also tested in
dose response in Hep3B cells by transfection.
IC50 (nM)
Free
Transfection
uptake
(RNAiMax)
AD-53063.1
1.60
0.03
AD-53001.1
2.27
0.01
AD-53015.1
2.90
0.02
AD-52953.1
2.94
0.03
AD-52986.1
3.30
0.03
AD-53024.1
3.42
0.02
AD-53033.1
3.42
0.02
AD-53027.1
3.84
0.01
AD-53030.1
3.90
0.03
AD-53080.1
4.08
0.04
AD-53073.1
4.20
0.05
AD-52965.1
4.63
ND
AD-53092.1
5.37
ND
AD-53132.1
5.54
ND
AD-52983.1
5.55
ND
AD-52954.1
5.67
ND
AD-52961.1
6.37
ND
AD-52994.1
6.43
ND
AD-53098.1
6.58
ND
AD-52970.1
6.71
ND
AD-53075.1
6.74
ND
AD-53086.1
7.08
ND
AD-52971.1
7.50
ND
AD-53064.1
8.33
ND
AD-53147.1
8.34
ND
AD-52969.1
8.86
ND
AD-53077.1
8.98
ND
AD-52981.1
9.44
ND
AD-52977.1
10.45
ND
AD-53071.1
11.19
ND
AD-52960.1
13.03
ND
AD-53095.1
21.31
ND
AD-53103.1
21.92
ND
TABLE 13
Results of single dose screen using sequences listed in Table 10.
STDEV
STDEV
STDEV
Duplex
10 nM
0.1 nM
0.025 nM
10 nM
0.1 nM
0.025 nM
AD-52719.1
0.01
0.60
0.35
0.000
0.093
0.002
AD-52717.1
0.02
0.31
0.32
0.001
0.014
0.008
AD-52713.1
0.02
0.37
0.36
0.001
0.011
0.007
AD-52711.1
0.03
0.22
0.23
0.005
0.011
0.009
AD-52718.1
0.03
0.31
0.39
0.000
0.025
0.023
AD-52687.1
0.03
0.37
0.38
0.005
0.020
0.002
AD-52699.1
0.03
0.25
0.21
0.002
0.011
0.002
AD-52679.1
0.03
0.51
0.24
0.345
0.008
AD-52689.1
0.03
0.44
0.42
0.000
0.039
0.002
AD-52700.1
0.03
0.56
0.57
0.005
0.044
0.020
AD-52637.1
0.04
0.27
0.23
0.001
0.003
0.005
AD-52730.1
0.04
0.61
0.59
0.005
0.053
0.014
AD-52725.1
0.04
0.62
0.61
0.002
0.027
0.012
AD-52688.1
0.04
0.23
0.20
0.006
0.012
0.011
AD-52661.1
0.04
0.61
0.25
0.001
0.449
0.009
AD-52667.1
0.04
0.28
0.22
0.004
0.018
0.013
AD-52665.1
0.04
0.43
0.48
0.007
0.019
0.009
AD-52638.1
0.04
0.28
0.25
0.000
0.016
0.027
AD-52724.1
0.05
0.86
0.76
0.001
0.055
0.011
AD-52705.1
0.05
0.74
0.65
0.004
0.022
0.016
AD-52708.1
0.05
0.53
0.52
0.001
0.034
0.013
AD-52659.1
0.05
0.56
0.48
0.000
0.000
0.033
AD-52678.1
0.05
0.53
0.53
0.002
0.034
0.000
AD-52670.1
0.05
0.35
0.33
0.002
0.009
0.003
AD-52695.1
0.05
0.63
0.67
0.001
0.012
0.013
AD-52704.1
0.05
0.55
0.53
0.002
0.005
0.034
AD-52683.1
0.05
0.36
0.28
0.002
0.021
0.011
AD-52673.1
0.05
0.22
0.19
0.023
0.010
0.002
AD-52721.1
0.05
0.60
0.53
0.003
0.006
0.029
AD-52710.1
0.05
0.56
0.40
0.007
0.073
0.000
AD-52714.1
0.05
0.40
0.51
0.000
0.016
0.003
AD-52686.1
0.05
0.57
0.60
0.003
0.014
0.000
AD-52645.1
0.05
0.62
0.59
0.004
0.030
0.003
AD-52662.1
0.05
0.55
0.52
0.002
0.030
0.008
AD-52720.1
0.05
0.50
0.46
0.003
0.007
0.011
AD-52654.1
0.05
0.29
0.36
0.008
0.037
0.014
AD-52680.1
0.06
0.48
0.41
0.001
0.019
0.026
AD-52723.1
0.06
0.84
0.76
0.001
0.041
0.004
AD-52726.1
0.06
0.72
0.66
0.003
0.028
0.016
AD-52701.1
0.06
0.67
0.39
0.001
0.003
0.002
AD-52694.1
0.06
0.68
0.59
0.004
0.040
0.012
AD-52685.1
0.06
0.30
0.25
0.002
0.013
0.016
AD-52728.1
0.06
0.80
0.79
0.005
0.043
0.015
AD-52676.1
0.06
0.68
0.67
0.002
0.023
0.029
AD-52639.1
0.06
0.47
0.45
0.000
0.005
0.007
AD-52722.1
0.06
0.81
0.93
0.005
0.004
0.027
AD-52682.1
0.06
0.87
0.73
0.009
0.038
0.014
AD-52660.1
0.07
0.69
0.68
0.002
0.014
0.017
AD-52709.1
0.07
0.89
0.82
0.001
0.013
0.020
AD-52643.1
0.07
0.27
0.24
0.006
0.016
0.012
AD-52696.1
0.07
0.53
0.46
0.003
0.026
0.007
AD-52657.1
0.08
0.60
0.58
0.008
0.030
0.006
AD-52706.1
0.08
0.84
0.78
0.001
0.021
0.019
AD-52653.1
0.08
0.41
0.45
0.057
0.004
0.029
AD-52656.1
0.08
0.65
0.50
0.004
0.022
0.012
AD-52693.1
0.09
0.61
0.62
0.007
0.021
0.018
AD-52692.1
0.09
0.54
0.52
0.023
0.018
0.033
AD-52674.1
0.10
0.79
0.64
0.001
0.008
0.028
AD-52648.1
0.10
0.67
0.53
0.002
0.013
0.028
AD-52651.1
0.10
0.84
0.73
0.000
0.000
0.007
AD-52641.1
0.10
0.62
0.50
0.004
0.172
0.002
AD-52707.1
0.10
0.92
0.81
0.001
0.018
0.032
AD-52671.1
0.11
0.87
0.84
0.005
0.034
0.025
AD-52650.1
0.12
0.88
0.94
0.007
0.013
0.041
AD-52642.1
0.12
0.90
0.76
0.015
0.022
0.004
AD-52675.1
0.13
0.94
0.89
0.001
0.018
0.044
AD-52647.1
0.13
0.80
0.79
0.031
0.008
0.023
AD-52716.1
0.14
0.61
0.69
0.010
0.060
0.013
AD-52649.1
0.14
0.31
0.29
0.136
0.020
0.006
AD-52677.1
0.16
1.01
0.72
0.059
0.040
0.007
AD-52697.1
0.16
0.86
0.77
0.012
0.021
0.015
AD-52715.1
0.17
0.90
0.89
0.005
0.009
0.022
AD-52691.1
0.18
0.93
0.88
0.004
0.036
0.017
AD-52698.1
0.20
0.97
0.87
0.010
0.028
0.000
AD-52672.1
0.20
0.70
0.66
0.170
0.014
0.019
AD-52712.1
0.29
0.92
0.90
0.007
0.036
0.004
AD-52690.1
0.30
0.95
0.85
0.115
0.032
0.004
AD-52640.1
0.30
1.04
0.91
0.018
0.046
0.013
AD-52684.1
0.31
0.90
0.94
0.014
0.018
0.014
AD-52666.1
0.32
1.04
0.91
0.013
0.005
0.004
AD-52703.1
0.32
1.02
0.96
0.016
0.015
0.005
AD-52729.1
0.33
1.02
0.87
0.032
0.020
0.008
AD-52668.1
0.35
0.94
0.90
0.029
0.046
0.026
AD-52681.1
0.57
1.00
0.99
0.003
0.034
0.039
AD-52702.1
0.72
1.02
0.92
0.658
0.060
0.014
AD-52727.1
0.73
1.03
0.91
0.004
0.065
0.027
AD-52663.1
0.78
1.05
0.96
0.027
0.010
0.005
AD-52669.1
0.91
0.91
0.94
0.004
0.049
0.032
AD-1955
0.95
0.84
0.95
0.005
0.021
0.019
AD-1955
0.97
1.07
1.03
0.000
0.021
0.015
AD-1955
1.01
1.08
1.01
0.035
0.011
0.005
mock
1.02
0.96
0.97
0.030
0.037
0.005
AD-1955
1.08
1.03
1.02
0.032
0.051
0.005
AD-52652.1
1.13
1.11
1.02
0.028
0.043
0.020
AD-52658.1
1.33
1.10
0.93
0.091
0.043
0.018
AD-52664.1
1.49
0.95
0.88
0.438
0.019
0.009
AD-52752.1
0.03
0.43
0.69
0.002
0.015
0.017
AD-52741.1
0.03
0.56
0.86
0.001
0.044
0.021
AD-52804.1
0.03
0.49
0.89
0.001
0.002
0.017
AD-52764.1
0.03
0.54
0.79
0.005
0.016
0.078
AD-52770.1
0.03
0.58
0.78
0.000
0.006
0.027
AD-52735.1
0.03
0.31
0.46
0.003
0.031
0.009
AD-52810.1
0.03
0.67
0.86
0.001
0.013
0.025
AD-52759.1
0.03
0.54
0.79
0.000
0.018
0.023
AD-52736.1
0.03
0.51
0.60
0.004
0.012
0.023
AD-52775.1
0.03
0.54
0.73
0.005
0.024
0.022
AD-52758.1
0.03
0.57
0.78
0.001
0.014
0.050
AD-52743.1
0.03
0.45
0.67
0.002
0.018
0.033
AD-52747.1
0.04
0.57
0.84
0.002
0.061
0.058
AD-52819.1
0.04
0.26
0.45
0.005
0.001
0.022
AD-52765.1
0.04
0.68
0.83
0.000
0.013
0.053
AD-52754.1
0.04
0.76
1.00
0.000
0.007
0.015
AD-52787.1
0.05
0.55
0.68
0.001
0.043
0.060
AD-52791.1
0.05
0.70
0.91
0.001
0.014
0.084
AD-52811.1
0.05
0.73
0.84
0.002
0.014
0.058
AD-52817.1
0.05
0.77
0.92
0.003
0.011
0.031
AD-52745.1
0.06
0.62
0.77
0.007
0.021
0.000
AD-52749.1
0.06
0.63
0.88
0.005
0.037
0.043
AD-52740.1
0.06
0.83
0.94
0.007
0.012
0.051
AD-52796.1
0.06
0.72
0.92
0.003
0.021
0.054
AD-52820.1
0.06
0.90
0.87
0.001
0.026
0.064
AD-52809.1
0.06
0.76
0.90
0.001
0.037
0.027
AD-52760.1
0.06
0.81
0.97
0.001
0.056
0.047
AD-52767.1
0.07
0.55
0.55
0.001
0.016
0.013
AD-52734.1
0.07
0.61
0.64
0.004
0.003
0.003
AD-52794.1
0.07
0.94
0.87
0.007
0.014
0.051
AD-52797.1
0.07
0.69
0.87
0.004
0.000
0.038
AD-52737.1
0.08
0.70
0.84
0.004
0.031
0.012
AD-52812.1
0.08
0.75
0.88
0.004
0.000
0.056
AD-52748.1
0.08
0.70
0.89
0.001
0.010
0.009
AD-52782.1
0.08
0.68
0.78
0.004
0.023
0.011
AD-52816.1
0.08
0.71
0.88
0.003
0.042
0.060
AD-52763.1
0.08
0.68
0.77
0.002
0.013
0.026
AD-52788.1
0.08
0.89
1.00
0.004
0.017
0.034
AD-52762.1
0.08
0.78
0.91
0.007
0.046
0.009
AD-52785.1
0.08
0.88
0.95
0.002
0.004
0.019
AD-52800.1
0.09
0.82
0.94
0.001
0.040
0.005
AD-52792.1
0.09
0.93
0.94
0.002
0.018
0.037
AD-52784.1
0.10
0.84
0.92
0.000
0.066
0.032
AD-52746.1
0.10
0.82
0.93
0.002
0.060
0.059
AD-52814.1
0.10
0.85
0.88
0.002
0.042
0.013
AD-52751.1
0.10
0.88
0.98
0.005
0.030
0.067
AD-52786.1
0.10
0.81
0.81
0.006
0.028
0.048
AD-52755.1
0.10
0.93
0.99
0.003
0.032
0.048
AD-52808.1
0.11
0.98
0.92
0.000
0.038
0.032
AD-52815.1
0.11
0.96
0.96
0.002
0.009
0.000
AD-52805.1
0.11
0.79
0.86
0.003
0.050
0.008
AD-52777.1
0.11
0.88
0.94
0.001
0.065
0.000
AD-52756.1
0.11
0.92
0.91
0.003
0.032
0.004
AD-52733.1
0.12
0.66
0.65
0.005
0.071
0.022
AD-52739.1
0.13
0.83
0.95
0.002
0.008
0.061
AD-52780.1
0.13
0.70
0.67
0.012
0.021
0.059
AD-52798.1
0.13
0.64
0.97
0.001
0.006
0.038
AD-52776.1
0.14
0.97
0.94
0.011
0.029
0.023
AD-52753.1
0.15
0.88
1.09
0.001
0.048
0.005
AD-52778.1
0.16
0.76
0.69
0.003
0.067
0.003
AD-52744.1
0.16
0.90
0.91
0.002
0.000
0.049
AD-52750.1
0.16
0.87
1.01
0.000
0.060
0.055
AD-52774.1
0.17
0.71
0.89
0.002
0.010
0.017
AD-52803.1
0.18
0.87
0.92
0.015
0.026
0.040
AD-52821.1
0.18
0.86
0.87
0.005
0.046
0.055
AD-52781.1
0.18
0.78
0.66
0.008
0.000
0.023
AD-52779.1
0.20
0.83
0.66
0.002
0.024
0.016
AD-52793.1
0.20
0.74
0.88
0.010
0.025
0.069
AD-52799.1
0.20
0.75
1.01
0.005
0.018
0.010
AD-52761.1
0.22
0.83
0.92
0.000
0.024
0.023
AD-52768.1
0.22
0.96
0.97
0.001
ND
0.028
AD-52757.1
0.23
1.02
0.95
0.018
0.040
0.042
AD-52806.1
0.24
0.96
0.87
0.011
0.084
0.055
AD-52771.1
0.25
0.92
0.98
0.010
0.018
0.048
AD-52802.1
0.30
0.95
1.00
0.010
0.019
0.005
AD-52731.1
0.30
0.85
0.75
0.001
0.067
0.022
AD-52813.1
0.30
1.07
0.98
0.001
0.109
0.014
AD-52742.1
0.31
0.95
1.03
0.005
0.028
0.056
AD-52766.1
0.35
0.97
1.00
0.010
0.024
0.044
AD-52732.1
0.41
0.79
0.73
0.004
0.016
0.039
AD-52773.1
0.43
0.99
0.92
0.004
0.029
0.022
AD-52772.1
0.43
1.00
1.02
0.006
0.000
0.065
AD-52822.1
0.44
0.68
0.81
0.004
0.010
0.016
AD-52783.1
0.45
0.66
0.76
0.009
0.036
0.019
AD-52789.1
0.50
0.68
0.78
0.010
0.053
0.004
AD-52795.1
0.50
0.82
0.69
0.000
0.080
0.054
AD-52801.1
0.54
0.70
0.79
0.018
0.038
0.035
AD-52807.1
0.57
0.76
0.93
0.006
0.011
0.032
AD-52769.1
0.76
0.97
0.92
0.015
0.085
0.045
AD-1955
0.90
0.96
1.04
0.018
0.165
0.010
AD-52818.1
0.92
1.03
0.92
0.009
0.010
0.063
AD-1955
1.01
0.90
0.96
0.005
0.031
0.019
AD-1955
1.05
1.09
1.00
0.046
0.085
0.005
AD-1955
1.05
1.07
1.00
0.010
0.031
0.039
mock
1.20
0.98
0.92
0.000
0.014
0.005
mock
1.25
0.99
1.00
0.006
0.005
0.034
TABLE 14
Results of a dose response screen
using a subset of sequences from Table 13.
A subset of active ANGPTL3 siRNAs
from Table 10 were tested by transfection in
Hep3B cells in dose response screens.
Duplex
IC50 (nM)
AD-52819.1
0.0036
AD-52667.1
0.0037
AD-52638.1
0.0048
AD-52673.1
0.0049
AD-52711.1
0.0050
AD-52661.1
0.0054
AD-52654.1
0.0058
AD-52637.1
0.0058
AD-52643.1
0.0060
AD-52685.1
0.0062
AD-52670.1
0.0064
AD-52679.1
0.0064
AD-52649.1
0.0066
AD-52683.1
0.0069
AD-52688.1
0.0071
AD-52717.1
0.0072
AD-52699.1
0.0073
AD-52714.1
0.0086
AD-52718.1
0.0088
AD-52735.1
0.0093
AD-52653.1
0.0102
AD-52687.1
0.0109
AD-52680.1
0.0120
AD-52713.1
0.0133
AD-52720.1
0.0143
AD-52639.1
0.0161
AD-52696.1
0.0163
AD-52662.1
0.0179
AD-52659.1
0.0180
AD-52710.1
0.0195
AD-52689.1
0.0216
AD-52787.1
0.0242
AD-52765.1
0.0318
TABLE 15
IDs of duplex pairs for which both an
unconjuaged and a GalNac-conjugated
version were synthesized and tested
These duplexes have the same
sequence and modification pattern.
GalNac
Unconjugated
conjugated
duplex
duplex
ID
ID
AD-52637.1
AD-52953.1
AD-52638.1
AD-52954.1
AD-52639.1
AD-52955.1
AD-52640.1
AD-52956.1
AD-52641.1
AD-52957.1
AD-52642.1
AD-52958.1
AD-52643.1
None
None
AD-52960.1
None
AD-52961.1
AD-52645.1
AD-52962.1
AD-52647.1
AD-52963.1
AD-52648.1
AD-52964.1
AD-52649.1
AD-52965.1
AD-52650.1
AD-52966.1
AD-52651.1
AD-52967.1
AD-52652.1
AD-52968.1
AD-52653.1
AD-52969.1
AD-52654.1
AD-52970.1
None
AD-52971.1
AD-52656.1
AD-52972.1
AD-52657.1
AD-52973.1
AD-52658.1
AD-52974.1
AD-52659.1
AD-52975.1
AD-52660.1
AD-52976.1
AD-52661.1
AD-52977.1
AD-52662.1
AD-52978.1
AD-52663.1
AD-52979.1
AD-52664.1
AD-52980.1
AD-52665.1
AD-52981.1
AD-52666.1
AD-52982.1
AD-52667.1
AD-52983.1
AD-52668.1
AD-52984.1
AD-52669.1
AD-52985.1
AD-52670.1
AD-52986.1
AD-52671.1
AD-52987.1
AD-52672.1
AD-52988.1
AD-52673.1
AD-52989.1
AD-52674.1
AD-52990.1
AD-52675.1
AD-52991.1
AD-52676.1
AD-52992.1
AD-52677.1
AD-52993.1
AD-52678.1
AD-52994.1
AD-52679.1
AD-52995.1
AD-52680.1
AD-52996.1
AD-52681.1
AD-52997.1
AD-52682.1
AD-52998.1
AD-52683.1
AD-52999.1
AD-52684.1
AD-53000.1
AD-52685.1
AD-53001.1
AD-52686.1
AD-53002.1
AD-52687.1
AD-53003.1
AD-52688.1
AD-53004.1
AD-52689.1
AD-53005.1
AD-52690.1
AD-53006.1
AD-52691.1
AD-53007.1
AD-52692.1
AD-53008.1
AD-52693.1
AD-53009.1
AD-52694.1
AD-53010.1
AD-52695.1
AD-53011.1
AD-52696.1
AD-53012.1
AD-52697.1
AD-53013.1
AD-52698.1
AD-53014.1
AD-52699.1
AD-53015.1
AD-52700.1
AD-53016.1
AD-52701.1
AD-53017.1
AD-52702.1
AD-53018.1
AD-52703.1
AD-53019.1
AD-52704.1
AD-53020.1
AD-52705.1
AD-53021.1
AD-52706.1
AD-53022.1
AD-52707.1
AD-53023.1
AD-52708.1
AD-53024.1
AD-52709.1
AD-53025.1
AD-52710.1
AD-53026.1
AD-52711.1
AD-53027.1
AD-52712.1
AD-53028.1
AD-52713.1
AD-53029.1
AD-52714.1
AD-53030.1
AD-52715.1
AD-53031.1
AD-52716.1
AD-53032.1
AD-52717.1
AD-53033.1
AD-52718.1
AD-53034.1
AD-52719.1
AD-53035.1
AD-52720.1
AD-53036.1
AD-52721.1
AD-53037.1
AD-52722.1
AD-53038.1
AD-52723.1
AD-53039.1
AD-52724.1
AD-53040.1
AD-52725.1
AD-53041.1
AD-52726.1
AD-53042.1
AD-52727.1
AD-53043.1
AD-52728.1
AD-53044.1
AD-52729.1
AD-53045.1
AD-52730.1
AD-53046.1
AD-52731.1
AD-53059.1
AD-52732.1
AD-53060.1
AD-52733.1
AD-53061.1
AD-52734.1
AD-53062.1
AD-52735.1
AD-53063.1
AD-52736.1
AD-53064.1
AD-52737.1
AD-53065.1
None
AD-53066.1
AD-52739.1
AD-53067.1
AD-52740.1
AD-53068.1
AD-52741.1
AD-53069.1
AD-52742.1
AD-53070.1
AD-52743.1
AD-53071.1
AD-52744.1
AD-53072.1
AD-52745.1
AD-53073.1
AD-52746.1
AD-53074.1
AD-52747.1
AD-53075.1
AD-52748.1
AD-53076.1
AD-52749.1
AD-53077.1
AD-52750.1
AD-53078.1
AD-52751.1
AD-53079.1
AD-52752.1
AD-53080.1
AD-52753.1
AD-53081.1
AD-52754.1
AD-53082.1
AD-52755.1
AD-53083.1
AD-52756.1
AD-53084.1
AD-52757.1
AD-53085.1
AD-52758.1
AD-53086.1
AD-52759.1
AD-53087.1
AD-52760.1
AD-53088.1
AD-52761.1
AD-53089.1
AD-52762.1
AD-53090.1
AD-52763.1
AD-53091.1
AD-52764.1
AD-53092.1
AD-52765.1
AD-53093.1
AD-52766.1
AD-53094.1
AD-52767.1
AD-53095.1
AD-52768.1
AD-53096.1
AD-52769.1
AD-53097.1
AD-52770.1
AD-53098.1
AD-52771.1
AD-53099.1
AD-52772.1
AD-53100.1
AD-52773.1
AD-53101.1
AD-52774.1
AD-53102.1
AD-52775.1
AD-53103.1
AD-52776.1
AD-53104.1
AD-52777.1
AD-53105.1
AD-52778.1
AD-53106.1
AD-52779.1
AD-53107.1
AD-52780.1
AD-53108.1
AD-52781.1
AD-53109.1
AD-52782.1
AD-53110.1
AD-52783.1
AD-53111.1
AD-52784.1
AD-53112.1
AD-52785.1
AD-53113.1
AD-52786.1
AD-53114.1
AD-52787.1
AD-53115.1
AD-52788.1
AD-53116.1
AD-52789.1
AD-53117.1
None
AD-53118.1
AD-52791.1
AD-53119.1
AD-52792.1
AD-53120.1
AD-52793.1
AD-53121.1
AD-52794.1
AD-53122.1
AD-52795.1
AD-53123.1
AD-52796.1
AD-53124.1
AD-52797.1
AD-53125.1
AD-52798.1
AD-53126.1
AD-52799.1
AD-53127.1
AD-52800.1
AD-53128.1
AD-52801.1
AD-53129.1
AD-52802.1
AD-53130.1
AD-52803.1
AD-53131.1
AD-52804.1
AD-53132.1
AD-52805.1
AD-53133.1
AD-52806.1
AD-53134.1
AD-52807.1
AD-53135.1
AD-52808.1
AD-53136.1
AD-52809.1
AD-53137.1
AD-52810.1
AD-53138.1
AD-52811.1
AD-53139.1
AD-52812.1
AD-53140.1
AD-52813.1
AD-53141.1
AD-52814.1
AD-53142.1
AD-52815.1
AD-53143.1
AD-52816.1
AD-53144.1
AD-52817.1
AD-53145.1
AD-52818.1
AD-53146.1
AD-52819.1
AD-53147.1
AD-52820.1
AD-53148.1
AD-52821.1
AD-53149.1
AD-52822.1
AD-53150.1
In Vivo Tests
Example 3
Test Articles
In vivo experiments were conducted using dsRNA sequences of the invention. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). The dsRNA sequence used as a negative control was luciferase-conjugated AD-48399B1 (“Luc”, sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). Also used as a negative control was GalNal-conjugated AD-1955 containing alternating 2′-methyl and 2′ fluoro modifications.
Experimental Procedure
The dsRNA sequences were tested in C57BL/6 (WT) and ob/ob mice. WT mice received five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5 or 20 mg/kg; and ob/ob mice received five daily doses of NPLs formulated with Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles were administered by subcutaneous injection according to the procedure shown in FIG. 1. Specifically, five daily doses of the test articles were administered on five consecutive days (day 0, 1, 2, 3 and 4), and blood samples were collected 5, 3 or 1 day prior to administration, as well as on days 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45 and 50 post-administration. The collected blood samples were used to measure the expression of ANGPTL3 protein using an ELISA assay. Levels of serum triglycerides (TGs), low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc) and total cholesterol (TC) were also measured using an Olympus Analyzer.
Results
Shown in FIG. 2, Panel A, are levels of murine ANGPTL3 (mANGPTL3, protein measured in WT mice after administration of control or ANG at 5 or 20 mg/kg.
Also shown in FIG. 2, Panel B are levels of mANGPTL3 protein measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that, for both WT and ob/ob mice, administration of ANG results in decreased levels of mANGPTL3 protein, as compared to controls.
Shown in FIG. 3, Panel A, are levels of LDL-c measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 3, Panel B are levels of LDL-c measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of LDL-c, particularly in ob/ob mice, as compared to controls.
Shown in FIG. 4, Panel A, are levels of triglycerides measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 4, Panel B are levels of triglycerides measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of tryglycerides, particularly, in ob/ob mice, as compared to controls.
Shown in FIG. 5, Panel A and B are levels of total cholesterol (TC) measured in WT and ob/ob mice, respectively, after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes a moderate decrease in TC levels in ob/ob mice, but not in WT mice. Similarly, administration of ANG causes a moderate decrease in HDL-c levels in ob/ob mice, but not in WT mice, as is shown in the graphs in FIG. 6.
Example 4
Test Article
The effect of a single injection of dsRNA sequence of the invention on the level of ANGPTL3 protein was tested. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). PBS was used as a negative control.
Experimental Procedure
The dsRNA sequences were tested in Human PCS Transgenic mouse characterized by liver-specific expression of full-length human PCSK9 gene. Human PCS transgenic mice were dosed with the AD-52981 or PBS using a single subcutaneous injection. The mice were divided into four groups, each group consisting of two males and two females. Each group received an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose of AD-52981. Blood samples were collected at day 1 and day 0 prior to dosing, and at 72 hours post dosing. ANGPTL3 protein levels were measured by ELISA and compared to levels at day 1 and day 0 prior to dosing.
Results
Shown in FIG. 7, are levels of murine ANGPTL3 protein (mANGPTL3) measured in Human PCS transgenic mice. The data shown is expressed relative to PBS control and represents an average for 2 males and 2 females in each group. Error bars represent standard deviation. The data indicates that administration of a single injection of AD-52981 reduces the levels of ANGPTL3 protein in the mice in a dose-dependent manner, with the dose of 60 mg/kg decreasing the levels of ANGPTL3 protein more than five-fold (see FIG. 7).
SEQUENCES
>gi|41327750|ref|NM_014495.2|
Homo sapiens angiopoietin-like 3
(ANGPTL3), mRNA
SEQ ID NO: 1
TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATT
GAAAATCAAGATAAAAATGTTCACAATTAAGCTCC
TTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGA
ATTGATCAAGACAATTCATCATTTGATTCTCTATC
TCCAGAGCCAAAATCAAGATTTGCTATGTTAGACG
ATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTG
GGACATGGTCTTAAAGACTTTGTCCATAAGACGAA
GGGCCAAATTAATGACATATTTCAAAAACTCAACA
TATTTGATCAGTCTTTTTATGATCTATCGCTGCAA
ACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAG
AAGAACTACATATAAACTACAAGTCAAAAATGAAG
AGGTAAAGAATATGTCACTTGAACTCAACTCAAAA
CTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCA
ACAAAAAGTGAAATATTTAGAAGAGCAACTAACTA
ACTTAATTCAAAATCAACCTGAAACTCCAGAACAC
CCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAA
ACAAGATAATAGCATCAAAGACCTTCTCCAGACCG
TGGAAGACCAATATAAACAATTAAACCAACAGCAT
AGTCAAATAAAAGAAATAGAAAATCAGCTCAGAAG
GACTAGTATTCAAGAACCCACAGAAATTTCTCTAT
CTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTT
CTTCAGTTGAATGAAATAAGAAATGTAAAACATGA
TGGCATTCCTGCTGAATGTACCACCATTTATAACA
GAGGTGAACATACAAGTGGCATGTATGCCATCAGA
CCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGA
TGTTATATCAGGTAGTCCATGGACATTAATTCAAC
ATCGAATAGATGGATCACAAAACTTCAATGAAACG
TGGGAGAACTACAAATATGGTTTTGGGAGGCTTGA
TGGAGAATTTTGGTTGGGCCTAGAGAAGATATACT
CCATAGTGAAGCAATCTAATTATGTTTTACGAATT
GAGTTGGAAGACTGGAAAGACAACAAACATTATAT
TGAATATTCTTTTTACTTGGGAAATCACGAAACCA
ACTATACGCTACATCTAGTTGCGATTACTGGCAAT
GTCCCCAATGCAATCCCGGAAAACAAAGATTTGGT
GTTTTCTACTTGGGATCACAAAGCAAAAGGACACT
TCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGG
TGGCATGATGAGTGTGGAGAAAACAACCTAAATGG
TAAATATAACAAACCAAGAGCAAAATCTAAGCCAG
AGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAAT
GGAAGGTTATACTCTATAAAATCAACCAAAATGTT
GATCCATCCAACAGATTCAGAAAGCTTTGAATGAA
CTGAGGCAAATTTAAAAGGCAATAATTTAAACATT
AACCTCATTCCAAGTTAATGTGGTCTAATAATCTG
GTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGA
TTTTTTTTATCTTAAAGTCACTGTCTATTTAAGAT
TAAACATACAATCACATAACCTTAAAGAATACCGT
TTACATTTCTCAATCAAAATTCTTATAATACTATT
TGTTTTAAATTTTGTGATGTGGGAATCAATTTTAG
ATGGTCACAATCTAGATTATAATCAATAGGTGAAC
TTATTAAATAACTTTTCTAAATAAAAAATTTAGAG
ACTTTTATTTTAAAAGGCATCATATGAGCTAATAT
CACAACTTTCCCAGTTTAAAAAACTAGTACTCTTG
TTAAAACTCTAAACTTGACTAAATACAGAGGACTG
GTAATTGTACAGTTCTTAAATGTTGTAGTATTAAT
TTCAAAACTAAAAATCGTCAGCACAGAGTATGTGT
AAAAATCTGTAATACAAATTTTTAAACTGATGCTT
CATTTTGCTACAAAATAATTTGGAGTAAATGTTTG
ATATGATTTATTTATGAAACCTAATGAAGCAGAAT
TAAATACTGTATTAAAATAAGTTCGCTGTCTTTAA
ACAAATGGAGATGACTACTAAGTCACATTGACTTT
AACATGAGGTATCACTATACCTTATT
>gi|297278846|ref|NM_001086114.2|
PREDICTED: Macaca mulatta
angiopoietin-like 3 (ANGPTL3), mRNA
SEQ ID NO: 2
ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAG
AAGAACACAGTTCCACGTTGCTTGAAATTGAAAAT
CAGGATAAAAATGTTCACAATTAAGCTCCTTCTTT
TTATTGTTCCTCTAGTTATTTCCTCCAGAATTGAC
CAAGACAATTCATCATTTGATTCTGTATCTCCAGA
GCCAAAATCAAGATTTGCTATGTTAGACGATGTAA
AAATTTTAGCCAATGGCCTCCTTCAGTTGGGACAT
GGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCA
AATTAATGACATATTTCAAAAACTCAACATATTTG
ATCAGTCTTTTTATGATCTATCACTGCAAACCAGT
GAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAAC
TACATATAAACTACAAGTCAAAAATGAAGAGGTAA
AGAATATGTCACTTGAACTCAACTCAAAACTTGAA
AGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAA
AGTGAAATATTTAGAAGAGCAACTAACTAACTTAA
TTCAAAATCAACCTGAAACTCCAGAACATCCAGAA
GTAACTTCACTTAAAAGTTTTGTAGAAAAACAAGA
TAATAGCATCAAAGACCTTCTCCAGACTGTGGAAG
AACAATATAAGCAATTAAACCAACAGCACAGTCAA
ATAAAAGAAATAGAAAATCAGCTCAGAATGACTAA
TATTCAAGAACCCACAGAAATTTCTCTATCTTCCA
AGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAG
CTGAATGAAATAAGAAATGTAAAACATGATGGCAT
TCCTGCTGATTGTACCACCATTTACAATAGAGGTG
AACATATAAGTGGCATGTATGCCATCAGACCCAGC
AACTCTCAAGTTTTTCATGTCTACTGTGATGTTGT
ATCAGGTAAAACCTGTCTAAGGAGAATAGATGGAT
CACAAAACTTCAATGAAACGTGGGAGAACTACAAA
TATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTT
GGGCCTAGAGAAGATATACTCCATAGTGAAGCAAT
CTAATTACGTTTTACGAATTGAGTTGGAAGACTGG
AAAGACAACAAACATTATATTGAATATTCTTTTTA
CTTGGGAAATCACGAAACCAACTATACGCTACATG
TAGTTAAGATTACTGGCAATGTCCCCAATGCAATC
CCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGA
TCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGA
GTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGT
GGAGAAAACAACCTAAATGGTAAATATAACAAACC
AAGAACAAAATCTAAGCCAGAGCGGAGAAGAGGAT
TATCCTGGAAGTCTCAAAATGGAAGGTTATACTCT
ATAAAATCAACCAAAATGTTGATCCATCCAACAGA
TTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAA
AAGGCAATAAATTAAACATTAAACTCATTCCAAGT
TAATGTGGTTTAATAATCTGGTATTAAATCCTTAA
GAGAAGGCTTGAGAAATAGATTTTTTTATCTTAAA
GTCACTGTCAATTTAAGATTAAACATACAATCACA
TAACCTTAAAGAATACCATTTACATTTCTCAATCA
AAATTCCTACAACACTATTTGTTTTATATTTTGTG
ATGTGGGAATCAATTTTAGATGGTCGCAATCTAAA
TTATAATCAACAGGTGAACTTACTAAATAACTTTT
CTAAATAAAAAACTTAGAGACTTTAATTTTAAAAG
TCATCATATGAGCTAATATCACAATTTTCCCAGTT
TAAAAAACTAGTTTTCTTGTTAAAACTCTAAACTT
GACTAAATAAAGAGGACTGATAATTATACAGTTCT
TAAATTTGTTGTAATATTAATTTCAAAACTAAAAA
TTGTCAGCACAGAGTATGTGTAAAAATCTGTAATA
TAAATTTTTAAACTGATGCCTCATTTTGCTACAAA
ATAATCTGGAGTAAATTTTTGATAGGATTTATTTA
TGAAACCTAATGAAGCAGGATTAAATACTGTATTA
AAATAGGTTCGCTGTCTTTTAAACAAATGGAGATG
ATGATTACTAAGTCACATTGACTTTAATATGAGGT
ATCACTATACCTTA
>gi|142388354|ref|NM_013913.3|
Mus musculus angiopoietin-like 3
(Angptl3), mRNA
SEQ ID NO: 3
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAA
TAATTGAGACAAAAAATGCACACAATTAAATTATT
CCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAG
TGGATCCAGACCTTTCATCATTTGATTCTGCACCT
TCAGAGCCAAAATCAAGATTTGCTATGTTGGATGA
TGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGG
GTCATGGACTTAAAGATTTTGTCCATAAGACTAAG
GGACAAATTAACGACATATTTCAGAAGCTCAACAT
ATTTGATCAGTCTTTTTATGACCTATCACTTCGAA
CCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGA
AGAACTACATCTACACTACAAGTTAAAAACGAGGA
GGTGAAGAACATGTCAGTAGAACTGAACTCAAAGC
TTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAA
CACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAA
CTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACC
CAGAAGTAACATCACTCAAAAGTTTTGTAGAACAG
CAAGACAACAGCATAAGAGAACTCCTCCAGAGTGT
GGAAGAACAGTATAAACAATTAAGTCAACAGCACA
TGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAG
ACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTC
TTCTAAATCAAGAGCACCAAGAACTACTCCCCCTC
TTCAACTGAACGAAACAGAAAATACAGAACAAGAT
GACCTTCCTGCCGACTGCTCTGCCGTTTATAACAG
AGGCGAACATACAAGTGGCGTGTACACTATTAAAC
CAAGAAACTCCCAAGGGTTTAATGTCTACTGTGAT
ACCCAATCAGGCAGTCCATGGACATTAATTCAACA
CCGGAAAGATGGCTCACAGGACTTCAACGAAACAT
GGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGAT
GGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGC
TATAGTCCAACAGTCTAACTACATTTTACGACTCG
AGCTACAAGACTGGAAAGACAGCAAGCACTACGTT
GAATACTCCTTTCACCTGGGCAGTCACGAAACCAA
CTACACGCTACATGTGGCTGAGATTGCTGGCAATA
TCCCTGGGGCCCTCCCAGAGCACACAGACCTGATG
TTTTCTACATGGAATCACAGAGCAAAGGGACAGCT
CTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGT
GGAATGACATATGTGGAGAAAACAACCTAAATGGA
AAATACAACAAACCCAGAACCAAATCCAGACCAGA
GAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCA
GAAAGCTCTATGCTATCAAATCATCCAAAATGATG
CTCCAGCCCACCACCTAAGAAGCTTCAACTGAACT
GAGACAAAATAAAAGATCAATAAATTAAATATTAA
AGTCCTCCCGATCACTGTAGTAATCTGGTATTAAA
ATTTTAATGGAAAGCTTGAGAATTGAATTTCAATT
AGGTTTAAACTCATTGTTAAGATCAGATATCACCG
AATCAACGTAAACAAAATTTATC
>gi|68163568|ref|NM_001025065.1|
Rattus norvegicus angiopoietin-like 3
(Angptl3), mRNA
SEQ ID NO: 4
GACGTTCCAAATTGCTTGAAATTGAATAATTGAAA
CAAAAATGCACACAATTAAGCTGCTCCTTTTTGTT
GTTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGA
CCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAA
AATCAAGATTTGCTATGTTGGATGATGTCAAAATT
TTAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCT
TAAAGATTTTGTCCATAAGACAAAGGGACAAATTA
ATGACATATTTCAGAAGCTCAACATATTTGATCAG
TGTTTTTATGACCTATCACTTCAAACCAATGAAAT
CAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACAT
CTAAACTACAAGTTAAAAACGAAGAGGTGAAGAAT
ATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCT
ACTGGAGGAGAAGATGGCGCTCCAACACAGAGTCA
GGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAG
AACCCGCCTGGGGCTCGGGAGCACCCAGAGGTAAC
GTCACTTAAAAGTTTTGTAGAACAGCAAGATAACA
GCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAA
TATAAACAACTAAGTCAACAGCACATTCAGATAAA
AGAAATAGAAAATCAGCTCAGAAAGACTGGCATTC
AAGAACCCACTGAAAATTCTCTTTATTCTAAACCA
AGAGCACCAAGAACTACTCCCCCTCTTCATCTGAA
GGAAGCAAAAAATATAGAACAAGATGATCTGCCTG
CTGACTGCTCTGCCATTTATAACAGAGGTGAACAT
ACAAGTGGCGTGTATACTATTAGACCAAGCAGCTC
TCAAGTGTTTAATGTCTACTGTGACACCCAATCAG
GCACTCCACGGACATTAATTCAACACCGGAAAGAT
GGCTCTCAAAACTTCAACCAAACGTGGGAAAACTA
CGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGA
TTTCCTTGCATCACTCACTTATCTGTTGATTTAAT
AGTATTAGTTGGGTGTGTTGACACAGGCCTGAGAC
CATAGCGCTTTTGGGCAAGGGGGGAGGAGGAGCAG
CAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCA
CACATTGATACTCCTTCTCGACATTAAGAATTATA
AATTAAGCAGCAATTATAAAATGGGCTGTGGAAAT
GTAACAATAAGCAAAAGCAGACCCCAGTCTTCATA
AAACTGATTGGTAAATATTATCCATGATAGCAACT
GCAATGATCTCATTGTACTTATCACTACTGCATGC
CTGCAGTATGCTTGTTGAAACTTAATTCTATAGTT
CATGGTTATCATAAGTCTTATTAAGGAACATAGTA
TACGCCATTGGCTCTAGTGAGGGGCCATGCTACAA
ATGAGCTGCAAAGATAGCAGTATAGAGCTCTTTCA
GTGATATCCTAAGCACAACGTAACACAGGTGAAAT
GGGCTGGAGGCACAGTTGTGGTGGAACACGCGGCC
AGCAGGACACTGGGACTGATCCCCAGCAGCACAAA
GAAAGTGATAGGAACACAGAGCGAGAGTTAGAAGG
GACAGGGTCACCGTCAGAGATACGGTGTCTAACTC
CTGCAACCCTACCTGTAATTATTCCATATTATAAA
CATATACTATATAACTGTGGGTCTCTGCATGTTCT
AGAATATGAATTCTATTTGATTGTAAAACAAAACT
ATAAAAATAAGTAAAAAAATAAAAAATAAACAGAT
ACTTAAAATCAAAAAAAAAAAAAAAAAAAAAAAAA
Reverse Complement of SEQ ID NO: 1
SEQ ID NO: 5
AATAAGGTATAGTGATACCTCATGTTAAAGTCAAT
GTGACTTAGTAGTCATCTCCATTTGTTTAAAGACA
GCGAACTTATTTTAATACAGTATTTAATTCTGCTT
CATTAGGTTTCATAAATAAATCATATCAAACATTT
ACTCCAAATTATTTTGTAGCAAAATGAAGCATCAG
TTTAAAAATTTGTATTACAGATTTTTACACATACT
CTGTGCTGACGATTTTTAGTTTTGAAATTAATACT
ACAACATTTAAGAACTGTACAATTACCAGTCCTCT
GTATTTAGTCAAGTTTAGAGTTTTAACAAGAGTAC
TAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCT
CATATGATGCCTTTTAAAATAAAAGTCTCTAAATT
TTTTATTTAGAAAAGTTATTTAATAAGTTCACCTA
TTGATTATAATCTAGATTGTGACCATCTAAAATTG
ATTCCCACATCACAAAATTTAAAACAAATAGTATT
ATAAGAATTTTGATTGAGAAATGTAAACGGTATTC
TTTAAGGTTATGTGATTGTATGTTTAATCTTAAAT
AGACAGTGACTTTAAGATAAAAAAAATCTATTTCT
CAAGCTTTCTCTTAAGGATTTAATACCAGATTATT
AGACCACATTAACTTGGAATGAGGTTAATGTTTAA
ATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAA
AGCTTTCTGAATCTGTTGGATGGATCAACATTTTG
GTTGATTTTATAGAGTATAACCTTCCATTTTGAGA
CTTCCAAGATAATCCTCTTCTCCTCTCTGGCTTAG
ATTTTGCTCTTGGTTTGTTATATTTACCATTTAGG
TTGTTTTCTCCACACTCATCATGCCACCACCAGCC
TCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTT
TTGCTTTGTGATCCCAAGTAGAAAACACCAAATCT
TTGTTTTCCGGGATTGCATTGGGGACATTGCCAGT
AATCGCAACTAGATGTAGCGTATAGTTGGTTTCGT
GATTTCCCAAGTAAAAAGAATATTCAATATAATGT
TTGTTGTCTTTCCAGTCTTCCAACTCAATTCGTAA
AACATAATTAGATTGCTTCACTATGGAGTATATCT
TCTCTAGGCCCAACCAAAATTCTCCATCAAGCCTC
CCAAAACCATATTTGTAGTTCTCCCACGTTTCATT
GAAGTTTTGTGATCCATCTATTCGATGTTGAATTA
ATGTCCATGGACTACCTGATATAACATCACAGTAG
ACATGAAAAACTTGAGAGTTGCTGGGTCTGATGGC
ATACATGCCACTTGTATGTTCACCTCTGTTATAAA
TGGTGGTACATTCAGCAGGAATGCCATCATGTTTT
ACATTTCTTATTTCATTCAACTGAAGAAAGGGAGT
AGTTCTTGGTGCTCTTGGCTTGGAAGATAGAGAAA
TTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGC
TGATTTTCTATTTCTTTTATTTGACTATGCTGTTG
GTTTAATTGTTTATATTGGTCTTCCACGGTCTGGA
GAAGGTCTTTGATGCTATTATCTTGTTTTTCTACA
AAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGG
AGTTTCAGGTTGATTTTGAATTAAGTTAGTTAGTT
GCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGA
ATTTTTTCTTCTAGGAGGCTTTCAAGTTTTGAGTT
GAGTTCAAGTGACATATTCTTTACCTCTTCATTTT
TGACTTGTAGTTTATATGTAGTTCTTCTCAGTTCC
TTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGA
TAGATCATAAAAAGACTGATCAAATATGTTGAGTT
TTTGAAATATGTCATTAATTTGGCCCTTCGTCTTA
TGGACAAAGTCTTTAAGACCATGTCCCAACTGAAG
GAGGCCATTGGCTAAAATTTTTACATCGTCTAACA
TAGCAAATCTTGATTTTGGCTCTGGAGATAGAGAA
TCAAATGATGAATTGTCTTGATCAATTCTGGAGGA
AATAACTAGAGGAACAATAAAAAGAAGGAGCTTAA
TTGTGAACATTTTTATCTTGATTTTCAATTTCAAG
CAACGTGGAACTGTTTTCTTCTGGAA
Reverse Complement of SEQ ID NO: 2
SEQ ID NO: 6
TAAGGTATAGTGATACCTCATATTAAAGTCAATGT
GACTTAGTAATCATCATCTCCATTTGTTTAAAAGA
CAGCGAACCTATTTTAATACAGTATTTAATCCTGC
TTCATTAGGTTTCATAAATAAATCCTATCAAAAAT
TTACTCCAGATTATTTTGTAGCAAAATGAGGCATC
AGTTTAAAAATTTATATTACAGATTTTTACACATA
CTCTGTGCTGACAATTTTTAGTTTTGAAATTAATA
TTACAACAAATTTAAGAACTGTATAATTATCAGTC
CTCTTTATTTAGTCAAGTTTAGAGTTTTAACAAGA
AAACTAGTTTTTTAAACTGGGAAAATTGTGATATT
AGCTCATATGATGACTTTTAAAATTAAAGTCTCTA
AGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCA
CCTGTTGATTATAATTTAGATTGCGACCATCTAAA
ATTGATTCCCACATCACAAAATATAAAACAAATAG
TGTTGTAGGAATTTTGATTGAGAAATGTAAATGGT
ATTCTTTAAGGTTATGTGATTGTATGTTTAATCTT
AAATTGACAGTGACTTTAAGATAAAAAAATCTATT
TCTCAAGCCTTCTCTTAAGGATTTAATACCAGATT
ATTAAACCACATTAACTTGGAATGAGTTTAATGTT
TAATTTATTGCCTTTTAAATTTGCCTCAGTTCATT
CAAAGCTTTCTGAATCTGTTGGATGGATCAACATT
TTGGTTGATTTTATAGAGTATAACCTTCCATTTTG
AGACTTCCAGGATAATCCTCTTCTCCGCTCTGGCT
TAGATTTTGTTCTTGGTTTGTTATATTTACCATTT
AGGTTGTTTTCTCCACACTCATCATGCCACCACCA
GCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTC
CTTTTGCTTTGTGATCCCAAGTAGAAAACACCAAA
TCTTTGTTTTCCGGGATTGCATTGGGGACATTGCC
AGTAATCTTAACTACATGTAGCGTATAGTTGGTTT
CGTGATTTCCCAAGTAAAAAGAATATTCAATATAA
TGTTTGTTGTCTTTCCAGTCTTCCAACTCAATTCG
TAAAACGTAATTAGATTGCTTCACTATGGAGTATA
TCTTCTCTAGGCCCAACCAGAATTCTCCATCAAGC
CTCCCGAAACCATATTTGTAGTTCTCCCACGTTTC
ATTGAAGTTTTGTGATCCATCTATTCTCCTTAGAC
AGGTTTTACCTGATACAACATCACAGTAGACATGA
AAAACTTGAGAGTTGCTGGGTCTGATGGCATACAT
GCCACTTATATGTTCACCTCTATTGTAAATGGTGG
TACAATCAGCAGGAATGCCATCATGTTTTACATTT
CTTATTTCATTCAGCTGAAGAAAGGGAGTAGTTCT
TGGTGCTCTTGGCTTGGAAGATAGAGAAATTTCTG
TGGGTTCTTGAATATTAGTCATTCTGAGCTGATTT
TCTATTTCTTTTATTTGACTGTGCTGTTGGTTTAA
TTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGT
CTTTGATGCTATTATCTTGTTTTTCTACAAAACTT
TTAAGTGAAGTTACTTCTGGATGTTCTGGAGTTTC
AGGTTGATTTTGAATTAAGTTAGTTAGTTGCTCTT
CTAAATATTTCACTTTTTGTTGAAGTAGAATTTTT
TCTTCTAGGAGGCTTTCAAGTTTTGAGTTGAGTTC
AAGTGACATATTCTTTACCTCTTCATTTTTGACTT
GTAGTTTATATGTAGTTCTTCTCAGTTCCTTTTCT
TCTTCTTTGATTTCACTGGTTTGCAGTGATAGATC
ATAAAAAGACTGATCAAATATGTTGAGTTTTTGAA
ATATGTCATTAATTTGGCCCTTAGTCTTATGGACA
AAGTCTTTAAGACCATGTCCCAACTGAAGGAGGCC
ATTGGCTAAAATTTTTACATCGTCTAACATAGCAA
ATCTTGATTTTGGCTCTGGAGATACAGAATCAAAT
GATGAATTGTCTTGGTCAATTCTGGAGGAAATAAC
TAGAGGAACAATAAAAAGAAGGAGCTTAATTGTGA
ACATTTTTATCCTGATTTTCAATTTCAAGCAACGT
GGAACTGTGTTCTTCTGGAAGCAGACCTAGACTTC
TTAACTCTATATAT
Reverse Complement of SEQ ID NO: 3
SEQ ID NO: 7
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAA
TAATTGAGACAAAAAATGCACACAATTAAATTATT
CCTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAG
TGGATCCAGACCTTTCATCATTTGATTCTGCACCT
TCAGAGCCAAAATCAAGATTTGCTATGTTGGATGA
TGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGG
GTCATGGACTTAAAGATTTTGTCCATAAGACTAAG
GGACAAATTAACGACATATTTCAGAAGCTCAACAT
ATTTGATCAGTCTTTTTATGACCTATCACTTCGAA
CCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGA
AGAACTACATCTACACTACAAGTTAAAAACGAGGA
GGTGAAGAACATGTCAGTAGAACTGAACTCAAAGC
TTGAGAGTCTGCTGGAAGAGAAGACAGCCCTTCAA
CACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAA
CTTAATTCTAAGCCCAGCTGGGGCTCAGGAGCACC
CAGAAGTAACATCACTCAAAAGTTTTGTAGAACAG
CAAGACAACAGCATAAGAGAACTCCTCCAGAGTGT
GGAAGAACAGTATAAACAATTAAGTCAACAGCACA
TGCAGATAAAAGAAATAGAAAAGCAGCTCAGAAAG
ACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTC
TTCTAAATCAAGAGCACCAAGAACTACTCCCCCTC
TTCAACTGAACGAAACAGAAAATACAGAACAAGAT
GACCTTCCTGCCGACTGCTCTGCCGTTTATAACAG
AGGCGAACATACAAGTGGCGTGTACACTATTAAAC
CAAGAAACTCCCAAGGGTTTAATGTCTACTGTGAT
ACCCAATCAGGCAGTCCATGGACATTAATTCAACA
CCGGAAAGATGGCTCACAGGACTTCAACGAAACAT
GGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGAT
GGAGAATTTTGGTTGGGCCTAGAGAAGATCTATGC
TATAGTCCAACAGTCTAACTACATTTTACGACTCG
AGCTACAAGACTGGAAAGACAGCAAGCACTACGTT
GAATACTCCTTTCACCTGGGCAGTCACGAAACCAA
CTACACGCTACATGTGGCTGAGATTGCTGGCAATA
TCCCTGGGGCCCTCCCAGAGCACACAGACCTGATG
TTTTCTACATGGAATCACAGAGCAAAGGGACAGCT
CTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGT
GGAATGACATATGTGGAGAAAACAACCTAAATGGA
AAATACAACAAACCCAGAACCAAATCCAGACCAGA
GAGAAGAAGAGGGATCTACTGGAGACCTCAGAGCA
GAAAGCTCTATGCTATCAAATCATCCAAAATGATG
CTCCAGCCCACCACCTAAGAAGCTTCAACTGAACT
GAGACAAAATAAAAGATCAATAAATTAAATATTAA
AGTCCTCCCGATCACTGTAGTAATCTGGTATTAAA
ATTTTAATGGAAAGCTTGAGAATTGAATTTCAATT
AGGTTTAAACTCATTGTTAAGATCAGATATCACCG
AATCAACGTAAACAAAATTTATC
Reverse Complement of SEQ ID NO: 4
SEQ ID NO: 8
TTTTTTTTTTTTTTTTTTTTTTTTTGATTTTAAGT
ATCTGTTTATTTTTTATTTTTTTACTTATTTTTAT
AGTTTTGTTTTACAATCAAATAGAATTCATATTCT
AGAACATGCAGAGACCCACAGTTATATAGTATATG
TTTATAATATGGAATAATTACAGGTAGGGTTGCAG
GAGTTAGACACCGTATCTCTGACGGTGACCCTGTC
CCTTCTAACTCTCGCTCTGTGTTCCTATCACTTTC
TTTGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCT
GGCCGCGTGTTCCACCACAACTGTGCCTCCAGCCC
ATTTCACCTGTGTTACGTTGTGCTTAGGATATCAC
TGAAAGAGCTCTATACTGCTATCTTTGCAGCTCAT
TTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTA
TACTATGTTCCTTAATAAGACTTATGATAACCATG
AACTATAGAATTAAGTTTCAACAAGCATACTGCAG
GCATGCAGTAGTGATAAGTACAATGAGATCATTGC
AGTTGCTATCATGGATAATATTTACCAATCAGTTT
TATGAAGACTGGGGTCTGCTTTTGCTTATTGTTAC
ATTTCCACAGCCCATTTTATAATTGCTGCTTAATT
TATAATTCTTAATGTCGAGAAGGAGTATCAATGTG
TGGCCCAGACTGGTCTTGAACTTTCAATTCACCTG
CTGCTCCTCCTCCCCCCTTGCCCAAAAGCGCTATG
GTCTCAGGCCTGTGTCAACACACCCAACTAATACT
ATTAAATCAACAGATAAGTGAGTGATGCAAGGAAA
TCACTTTACCATCAAGCCTCCCAAAACCCTTTTCG
TAGTTTTCCCACGTTTGGTTGAAGTTTTGAGAGCC
ATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGC
CTGATTGGGTGTCACAGTAGACATTAAACACTTGA
GAGCTGCTTGGTCTAATAGTATACACGCCACTTGT
ATGTTCACCTCTGTTATAAATGGCAGAGCAGTCAG
CAGGCAGATCATCTTGTTCTATATTTTTTGCTTCC
TTCAGATGAAGAGGGGGAGTAGTTCTTGGTGCTCT
TGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTT
GAATGCCAGTCTTTCTGAGCTGATTTTCTATTTCT
TTTATCTGAATGTGCTGTTGACTTAGTTGTTTATA
TTGTTCTTCCACACTCTGGAGGAGTTCTCTTATGC
TGTTATCTTGCTGTTCTACAAAACTTTTAAGTGAC
GTTACCTCTGGGTGCTCCCGAGCCCCAGGCGGGTT
CTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCC
TGACTCTGTGTTGGAGCGCCATCTTCTCCTCCAGT
AGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACAT
ATTCTTCACCTCTTCGTTTTTAACTTGTAGTTTAG
ATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTG
ATTTCATTGGTTTGAAGTGATAGGTCATAAAAACA
CTGATCAAATATGTTGAGCTTCTGAAATATGTCAT
TAATTTGTCCCTTTGTCTTATGGACAAAATCTTTA
AGACCATGACCCAGCTGCAGGAGGCCATTGGCTAA
AATTTTGACATCATCCAACATAGCAAATCTTGATT
TTGGCTCTGACGGTACAGAATCAAATGGCGAAAGG
TCTGGATCAACTCTGGACGAAATTACTAGAGGAAC
AACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTG
TTTCAATTATTCAATTTCAAGCAATTTGGAACGTC
Macaca fascicularis angiopoietin-
like 3 (Angptl3), mRNA
SEQ ID NO: 9
GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCT
TCCAGAAGAACACAGTTCCACGCTGCTTGAAATTG
AAAATCAGGATAAAAATGTTCACAATTAAGCTCCT
TCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAA
TTGACCAAGACAATTCATCATTTGATTCTGTATCT
CCAGAGCCAAAATCAAGATTTGCTATGTTAGACGA
TGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGG
GACATGGTCTTAAAGACTTTGTCCATAAGACTAAG
GGCCAAATTAATGACATATTTCAAAAACTCAACAT
ATTTGATCAGTCTTTTTATGATCTATCACTGCAAA
CCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGA
AGAACTACATATAAACTACAAGTCAAAAATGAAGA
GGTAAAGAATATGTCACTTGAACTCAACTCAAAAC
TTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAA
CAAAAAGTGAAATATTTAGAAGAGCAACTAACTAA
CTTAATTCAAAATCAACCTGCAACTCCAGAACATC
CAGAAGTAACTTCACTTAAAAGTTTTGTAGAAAAA
CAAGATAATAGCATCAAAGACCTTCTCCAGACTGT
GGAAGAACAATATAAGCAATTAAACCAACAGCATA
GTCAAATAAAAGAAATAGAAAATCAGCTCAGAATG
ACTAATATTCAAGAACCCACAGAAATTTCTCTATC
TTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTC
TTCAGCTGAATGAAATAAGAAATGTAAAACATGAT
GGCATTCCTGCTGATTGTACCACCATTTACAATAG
AGGTGAACATATAAGTGGCACGTATGCCATCAGAC
CCAGCAACTCTCAAGTTTTTCATGTCTACTGTGAT
GTTGTATCAGGTAGTCCATGGACATTAATTCAACA
TCGAATAGATGGATCACAAAACTTCAATGAAACGT
GGGAGAACTACAAATATGGTTTCGGGAGGCTTGAT
GGAGAATTCTGGTTGGGCCTAGAGAAGATATACTC
CATAGTGAAGCAATCTAATTACGTTTTACGAATTG
AGTTGGAAGACTGGAAAGACAACAAACATTATATT
GAATATTCTTTTTACTTGGGAAATCACGAAACCAA
CTATACGCTACATGTAGTTAAGATTACTGGCAATG
TCCCCAATGCAATCCCGGAAAACAAAGATTTGGTG
TTTTCTACTTGGGATCACAAAGCAAAAGGACACTT
CAGCTGTCCAGAGAGTTATTCAGGAGGCTGGTGGT
GGCATGATGAGTGTGGAGAAAACAACCTAAATGGT
AAATATAACAAACCAAGAACAAAATCTAAGCCAGA
GCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATG
GAAGGTTATACTCTATAAAATCAACCAAAATGTTG
ATCCATCCAACAGATTCAGAAAGCTTTGAATGAAC
TGAGGCAAATTTAAAAGGCAATAAATTAAACATTA
AACTCATTCCAAGTTAATGTGGTTTAATAATCTGG
TATTAAATCCTTAAGAGAAGGCTTGAGAAATAGAT
TTTTTTATCTTAAAGTCACTGTCAATTTAAGATTA
AACATACAATCACATAACCTTAAAGAATACCATTT
ACATTTCTCAATCAAAATTCTTACAACACTATTTG
TTTTATATTTTGTGATGTGGGAATCAATTTTAGAT
GGTCGCAATCTAAATTATAATCAACAGGTGAACTT
ACTAAATAACTTTTCTAAATAAAAAACTTAGAGAC
TTTAATTTTAAAAGTCATCATATGAGCTAATGTCA
CAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTT
AAAACTCTAAACTTGACTAAATAAAGAGGACTGAT
AATTATACAGTTCTTAAATTTGTTGTAATATTAAT
TTCAAAACTAAAAATTGTCAGCACAGAGTATGTGT
AAAAATCTGTAATATAAATTTTTAAACTGATGCCT
CATTTTGCTACAAAATAATCTGGAGTAAATTTTTG
ATAGGATTTATTTATGAAACCTAATGAAGCAGGAT
TAAATACTGTATTAAAATAGGTTCGCTGTCTTTTA
AACAAATGGAGATGATGATTACTAAGTCACATTGA
CTTTAATATGAGGTATCACTATACCTTAACATATT
TGTTAAAACGTATACTGTATACATTTTGTGT
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17531900
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alnylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 20th, 2022 03:05PM
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Apr 20th, 2022 03:05PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Apr 19th, 2022 12:00AM
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Nov 22nd, 2021 12:00AM
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https://www.uspto.gov?id=US11306315-20220419
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Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof
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The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.
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11306315
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1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), comprising a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the complement of nucleotides 244-307 of SEQ ID NO:1,
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, an abasic nucleotide, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
2. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), wherein the dsRNA agent comprises a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the complement of nucleotides 256-306 of SEQ ID NO:1,
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, an abasic nucleotide, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
3. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), wherein the dsRNA agent comprises a sense strand and an antisense strand,
wherein the antisense strand comprises at least 17 contiguous nucleotides which differ by no more than three nucleotides from the complement of nucleotides 266-299 of SEQ ID NO:1,
wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides,
wherein at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, an abasic nucleotide, and a 2′-amino modified nucleotide, and
wherein a ligand comprising an N-acetylgalactosamine (GalNAc) derivative is conjugated to at least one strand of the dsRNA agent.
4. The dsRNA agent of claim 1, wherein each strand is independently 19-25 nucleotides in length.
5. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
6. The dsRNA of claim 1, wherein the GalNAc (N-acetylgalactosamine) derivative is attached through a bivalent or trivalent branched linker.
7. The dsRNA agent of claim 1, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of
(SEQ ID NO: 312)
5′-AUCAGUCUUUUUAUGAUCUAU-3′
and
(SEQ ID NO: 497)
5′-AUAGAUCAUAAAAAGACUGAUCA-3′;
(SEQ ID NO: 403)
5′-GAUCAGUCUUUUUAUGAUCUA-3′
and
(SEQ ID NO: 588)
5′-UAGAUCAUAAAAAGACUGAUCAA-3′;
(SEQ ID NO: 61)
5′-AUUUGAUCAGUCUUUUUAU-3′
and
(SEQ ID NO: 123)
5′-AUAAAAAGACUGAUCAAAU-3′;
(SEQ ID NO: 16)
5′-UAUUUGAUCAGUCUUUUUA-3′
and
(SEQ ID NO: 78)
5′-UAAAAAGACUGAUCAAAUA-3′;
(SEQ ID NO: 387)
5′-CAUAUUUGAUCAGUCUUUUUA-3′
and
(SEQ ID NO: 572)
5′-UAAAAAGACUGAUCAAAUAUGUU-3′;
(SEQ ID NO: 287)
5′-ACAUAUUUGAUCAGUCUUUUU-3′
and
(SEQ ID NO: 472)
5′-AAAAAGACUGAUCAAAUAUGUUG-3′;
(SEQ ID NO: 303)
5′-AACAUAUUUGAUCAGUCUUUU-3′
and
(SEQ ID NO: 488)
5′-AAAAGACUGAUCAAAUAUGUUGA-3′;
(SEQ ID NO: 39)
5′-ACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 101)
5′-AAAGACUGAUCAAAUAUGU -3′;
(SEQ ID NO: 294)
5′-CAACAUAUUUGAUCAGUCUUU-3′
and
(SEQ ID NO: 479)
5′-AAAGACUGAUCAAAUAUGUUGAG-3′;
(SEQ ID NO: 358)
5′-UCAACAUAUUUGAUCAGUCUU-3′
and
(SEQ ID NO: 543)
5′-AAGACUGAUCAAAUAUGUUGAGU-3′;
(SEQ ID NO: 64)
5′-CAACAUAUUUGAUCAGUCU-3′
and
(SEQ ID NO: 126)
5′-AGACUGAUCAAAUAUGUUG-3′;
(SEQ ID NO: 321)
5′-CAAAAACUCAACAUAUUUGAU-3′
and
(SEQ ID NO: 506)
5′-AUCAAAUAUGUUGAGUUUUUGAA-3′;
(SEQ ID NO: 351)
5′-UGACAUAUUUCAAAAACUCAA-3′
and
(SEQ ID NO: 536)
5′-UUGAGUUUUUGAAAUAUGUCAUU-3′;
(SEQ ID NO: 352)
5′-AAAUUAAUGACAUAUUUCAAA-3′
and
(SEQ ID NO: 537)
5′-UUUGAAAUAUGUCAUUAAUUUGG-3′;
(SEQ ID NO: 285)
5′-GGCCAAAUUAAUGACAUAUUU-3′
and
(SEQ ID NO: 470)
5′-AAAUAUGUCAUUAAUUUGGCCCU-3′;
and
(SEQ ID NO: 417)
5′-GGGCCAAAUUAAUGACAUAUU-3′
and
(SEQ ID NO: 602)
5′-AAUAUGUCAUUAAUUUGGCCCUU-3′.
8. A cell containing the dsRNA agent of claim 1.
9. A pharmaceutical composition for inhibiting expression of an ANGPTL3 gene, comprising the dsRNA agent of claim 1.
10. The pharmaceutical composition of claim 9, wherein the dsRNA agent is present in a buffered solution.
11. A method of inhibiting ANGPTL3 expression in a cell, the method comprising:
(a) contacting the cell with the dsRNA agent of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
12. The method of claim 11, wherein the cell is within a subject.
13. A method of inhibiting the expression of ANGPTL3 in a subject, the method comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby inhibiting the expression of ANGPTL3 in the subject.
14. A method of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating the subject.
15. The method of claim 14, wherein the disorder is a disorder of lipid metabolism.
16. The method of claim 14, wherein the disorder is selected from the group consisting of hypertriglyceridemia, obesity, hyperlipidemia, atherosclerosis, diabetes, cardiovascular disease, and coronary artery disease.
17. The method of claim 14, further comprising administering an additional therapeutic to the subject.
18. The method of claim 17, wherein the additional therapeutic is a statin.
19. The method of claim 14, wherein the dsRNA agent is administered at a dose of about 0.5 mg/kg to about 50 mg/kg.
20. The method of claim 14, wherein the administration of the dsRNA agent to the subject causes a decrease in one or more serum lipid and/or a decrease in ANGPTL3 protein accumulation.
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RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 17/089,854, filed on Nov. 5, 2020, which is a continuation of U.S. patent application Ser. No. 16/411,261, filed on May 14, 2019, now U.S. Pat. No. 10,934,545, issued on Mar. 2, 2021, which is a continuation of U.S. patent application Ser. No. 15/683,999, filed on Aug. 23, 2017, now U.S. Pat. No. 10,337,010, issued on Jul. 2, 2019, which is a continuation of U.S. patent application Ser. No. 15/068,912 filed on Mar. 14, 2016, now U.S. Pat. No. 9,771,591, issued on Sep. 26, 2017, which is a continuation of U.S. patent application Ser. No. 14/132,999 filed on Dec. 18, 2013, now U.S. Pat. No. 9,322,018, issued on Apr. 26, 2016, which is a 35 U.S.C. 111(a) continuation application, which claims priority to PCT/US2012/043378, filed on Jun. 20, 2012, U.S. Provisional Application No. 61/499,620, filed on Jun. 21, 2011, and to U.S. Provisional Application No. 61/638,288, filed on Apr. 25, 2012. The entire contents of each of the foregoing applications are hereby incorporated herein by reference.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Nov. 19, 2021, is named 121301_00311_SL.txt and is 444,512 bytes in size.
BACKGROUND OF THE INVENTION
Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulates lipid metabolism and that is predominantly expressed in the liver (Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activities of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and of endothelial lipase (EL), which hydrolyzes high density lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese, KK/Snk mice, a reduction in ANGPTL3 expression has a protective effect against hyperlipidemia and artherosclerosis by promoting the clearance of triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223). Human ANGPTL3 plasma concentrations positively correlate with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).
Disorders of lipid metabolism can lead to elevated levels of serum lipids, such as triglycerides and/or cholesterol. Elevated serum lipids are strongly associated with high blood pressure, cardiovascular disease, diabetes and other pathologic conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder that is characterized by high blood levels of triglycerides. It has been associated with atherosclerosis, even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia can also lead to pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder that is characterized by elevated levels of any one or all lipids and/or lipoproteins in the blood. Current treatments for disorders of lipid metabolism, including dieting, exercise and treatment with statins and other drugs, are not always effective. Accordingly, there is a need in the art for alternative treatments for subjects having disorders of lipid metabolism.
SUMMARY OF THE INVENTION
The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPL3 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ANGPL3 gene, e.g., a subject suffering or prone to suffering from a disorder of lipid metabolism, such as a subject suffering or prone to suffering from hyperlipidemia or hypertriglyceridemia.
Accordingly, in one aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.
In another aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.
In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of AD-53063.1, AD-53001.1, AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 of Tables 7 and 8.
In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length.
In one embodiment, each strand of a dsRNA is no more than 30 nucleotides in length.
At least one strand of a dsRNA may comprise a 3′ overhang of at least 1 nucleotide or at least 2 nucleotides.
In certain embodiments, a dsRNA further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.
In some embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is
In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic
In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.
In one embodiment, the region of complementarity of a dsRNA consists of one of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, a dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10.
In another aspect, the present invention provides a cell, e.g., a hepatocyte, containing a dsRNA of the invention.
In yet another aspect, the present invention provides a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage. The region of complementarity may be least 15 nucleotides in length or 19 to 21 nucleotides in length.
In a further aspect, the present invention provides a cell comprising a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.
In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising a dsRNA or vector of the invention.
In one embodiment, the pharmaceutical composition comprises a lipid formulation, such as a MC3, SNALP or XTC formulation.
In another aspect, the present invention provides methods of inhibiting ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA or a vector of the invention, and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
The cell may be within a subject, such as a human subject, for example a human subject suffering from a disorder of lipid metabolism, e.g., hyperlipidemia or hypertriglyceridemia.
In one embodiment of the methods of the invention, ANGPTL3 expression is inhibited by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby treating the subject.
The disorder may be disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia
In one embodiment, the administration of the dsRNA to the subject causes a decrease in the level of a serum lipid, triglycerides, cholesterol and/or free fatty acids; and/or a decrease in ANGPTL3 protein accumulation. In one embodiment, administration of the dsRNA to the subject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, IDL-C and/or total cholesterol.
In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another aspect, the present invention provides methods of inhibiting the expression of ANGPTL3 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby inhibiting the expression of ANGPTL3 in the subject.
In yet another aspect, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method of inhibiting expression of ANGPTL3 gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the ANGPTL3 in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of the experimental procedure used for in vivo tests described in Example 2.
FIG. 2A is a graph showing measured levels of ANGPTL3 protein in WT mice after treatment with the indicated iRNA or a control.
FIG. 2B is a graph showing measured levels of ANGPTL3 proten in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 3A is a graph showing measured levels of LDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 3B is a graph showing measured levels of LDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 4A is a graph showing measured levels of triglycerides in WT mice after treatment with the indicated iRNA or a control.
FIG. 4B is a graph showing measured levels of triglycerides in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 5A is a graph showing measured levels of total cholesterol (TC) in WT mice after treatment with the indicated iRNA or a control.
FIG. 5B is a graph showing measured levels of total cholesterol (TC) in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 6A is a graph showing measured levels of HDL-c in WT mice after treatment with the indicated iRNA or a control.
FIG. 6B is a graph showing measured levels of HDL-c in ob/ob mice after treatment with the indicated iRNA or a control.
FIG. 7 is a graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or a control.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPTL3gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ANGPTL3 gene. The use of these iRNAs enables the targeted degradation of mRNAs of an ANGPTL3 gene in mammals. Very low dosages of ANGPTL3 iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of an ANGPTL3 gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of expression of an ANGPTL3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of an ANGPTL3 protein, such as a subject having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.
The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ANGPTL3 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.
I. Definitions
In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.
The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.
The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. The term “ANGPTL3” refers to an angiopoietin like protein 3 having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3. The term encompasses full-length unprocessed precursor forms of ANGPTL3 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the fibrinogen-like domain. The sequence of a human ANGPTL3 mRNA transcript can be found at, for example, GenBank Accession No. GI: 41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesus ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 142388354 (NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 68163568 (NM_001025065.1; SEQ ID NO:4).
The term“ANGPTL3” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the ANGPTL3 gene, such as a single nucleotide polymorphism in the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the ANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039; rs192778191; rs192764027; rs192528948; rs191931953; rs191293319; rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3 gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3gene.
The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of ANGPTL3 in a cell, e.g., a cell within a subject, such as a mammalian subject.
In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an ANGPTL3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.
In another aspect, the RNAi agent is a single-stranded antisense RNA molecule. An antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule may be about 13 to about 30 nucleotides in length and have a sequence that is complimentary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.
In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an ANGPTL3 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.
The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.
As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an ANGPTL3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.
The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a part of an ANGPTL3mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ANGPTL3.
In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.
The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.
The phrase “inhibiting expression of an ANGPTL3,” as used herein, includes inhibition of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 gene that encode an ANGPTL3 protein.
“Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.
The expression of an ANGPTL3 gene may be assessed based on the level of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of an ANGPTL3 may also be assessed indirectly based on the levels of a serum lipid, a triglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).
In one embodiment, at least partial suppression of the expression of an ANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNA which can be isolated from or detected in a first cell or group of cells in which an ANGPTL3 gene is transcribed and which has or have been treated such that the expression of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:
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100
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The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.
Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.
In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.
The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817, the entire contents of which are hereby incorporated herein by reference. Examples of “SNALP” formulations are described below.
As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human having a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as lowering levels of triglycerides in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a disorder of lipid metabolism, such as, e.g., a decrease in the size of eruptive xanthomas. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.
By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., high triglyceride levels or eruptive xanthoma. The likelihood of developing a high tryglyceride levels or eruptive xanthoma is reduced, for example, when an individual having one or more risk factors for a high tryglyceride levels or eruptive xanthoma either fails to develop high tryglyceride levels or eruptive xanthoma or develops high tryglyceride levels or eruptive xanthoma with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition i (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.
As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e.g., a lipoprotein complex. Non-limiting examples of serum lipids may include triglycerides and cholesterol, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).
As used herein, a “disorder of lipid metabolism” refers to any disorder associated with or caused by a disturbance in lipid metabolism. For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, or an acquired disorder, such as a disorder acquired as a result of a diet or intake of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrome, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).
Cardiovascular diseases associated with disorders of lipid metabolism are also considered “disorders of lipid metabolism”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke.
Disorders related to body weight are also considered “disorders of lipid metabolism”, as defined herein. Such disorders may include obesity, metabolic syndrome including independent components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss.
Blood sugar disorders are further considered “disorders of lipid metabolism”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of lipid metabolism may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.
“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a disorder of lipid metabolism, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.
“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a disorder of lipid metabolism, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.
A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.
II. iRNAs of the Invention
Described herein are iRNAs which inhibit the expression of an ANGPTL3 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a disorder of lipid metabolism, e.g., familial hyperlipidemia. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ANGPTL3gene, The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, a primate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.
A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an ANGPTL3gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.
In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ANGPTL3 expression is not generated in the target cell by cleavage of a larger dsRNA.
A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.
A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.
iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.
In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in Tables 2, 3, 7, 8, 9 and 10, and the corresponding antisense strand of the sense strand is selected from the group of sequences of Tables 2, 3, 7, 8, 9 and 10. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an ANGPTL3gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in Tables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.
The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 7, 8, 9 and 10, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2, 3, 7, 8, 9 and 10 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 2, 3, 7, 8, 9 and 10, and differing in their ability to inhibit the expression of an ANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.
In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify a site(s) in an ANGPTL3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 7, 8, 9 and 10 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ANGPTL3gene.
While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and 10 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 7, 8, 9 and 10, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.
An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an ANGPTL3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ANGPTL3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ANGPTL3 gene is important, especially if the particular region of complementarity in an ANGPTL3 gene is known to have polymorphic sequence variation within the population.
III. Modified iRNAs of the Invention
In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.
In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2-, —CH2—N(CH3)—O—CH2-[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2) nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.
An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.
Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.
IV. iRNAs Conjugated to Ligands
Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.
Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.
The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
A. Lipid Conjugates
In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).
B. Cell Permeation Agents
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
C. Carbohydrate Conjugates
In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).
In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as
In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:
Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,
(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.
In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.
D. Linkers
In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.
The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.
A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
i. Redox Cleavable Linking Groups
In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.
ii. Phosphate-based cleavable linking groups In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.
iii. Acid Cleavable Linking Groups
In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
iv. Ester-based linking groups In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.
v. Peptide-Based Cleaving Groups
In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.
In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,
when one of X or Y is an oligonucleotide, the other is a hydrogen.
In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.
In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):
wherein:
q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;
P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;
Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O);
R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)—CH(Ra)—NH—, CO, CH═N—O,
or heterocyclyl;
L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):
wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.
Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II_VII, XI, X, and XIII
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.
“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
IV. Delivery of an iRNA of the Invention
The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against
ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
A. Vector Encoded iRNAs of the Invention
iRNA targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., (1993) Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood 83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; and Wang et al., (1995) Gene Therapy 2:775-783. A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
V. Pharmaceutical Compositions of the Invention
The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, such as hypertriglyceridemia.
Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.
The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a ANGPTL3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on ANGPTL3 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of lipid metabolism that would benefit from reduction in the expression of ANGPTL3. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of an LDL receptor (LDLR−/−mouse; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced artherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).
The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
A. iRNA Formulations Comprising Membranous Molecular Assemblies
An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle.
As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.
A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.
Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).
Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P.Pharma. Sci., 4(6):466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85: 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.
Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.
Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.
Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.
A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.
B. Nucleic Acid Lipid Particles
iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid
DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.
The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N—(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N—(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNPO1 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
LNPO1 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary lipid-dsRNA formulations are described in the table below.
cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid
conjugate
Ionizable/Cationic Lipid
Lipid:siRNA ratio
SNALP-1
12-Dilinolenyloxy-N,N-dimethylaminopropane
DLinDMA/DPPC/Cholesterol/PEG-
(DLinDMA)
cDMA
(57.1/7.1/34.4/1.4)
lipid:siRNA ~7:1
2-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DPPC/Cholesterol/PEG-
dioxolane (XTC)
cDMA
57.1/7.1/34.4/1.4
lipid:siRNA ~7:1
LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG
dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA ~6:1
LNP06
2,2-Dilinoleyl-4-dimethylaminoethy1-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG
dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA ~11:1
LNP07
2,2-Dilinoleyl-4-dimethylaminoethy1-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG
dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA ~6:1
LNP08
2,2-Dilinoleyl-4-dimethylaminoethy1-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG
dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA ~11:1
LNP09
2,2-Dilinoley1-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG
dioxolane (XTC)
50/10/38.5/1.5
Lipid:siRNA 10:1
LNP10
(3aR,5s,6aS)-N,N-dimethy1-2,2-di((9Z,12Z)-
ALN100/DSPC/Cholesterol/PEG-
octadeca-9,12-dienyl)tetrahydro-3aH-
DMG
cyclopenta[d][1,3]dioxo1-5-amine (ALN100)
50/10/38.5/1.5
Lipid:siRNA 10:1
LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-
MC-3/DSPC/Cholesterol/PEG-
tetraen-19-yl 4-(dimethylamino)butanoate
DMG
(MC3)
50/10/38.5/1.5
Lipid:siRNA 10:1
LNP12
1,1′-(2-(4-(24(2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-
hydroxydodecyl)amino)ethyl)(2-
DMG
hydroxydodecyl)amino)ethyl)piperazin-1-
50/10/38.5/1.5
yl)ethylazanediyl)didodecan-2-ol (Tech G1)
Lipid:siRNA 10:1
LNP13
XTC
XTC/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 33:1
LNP14
MC3
MC3/DSPC/Chol/PEG-DMG
40/15/40/5
Lipid:siRNA: 11:1
LNP15
MC3
MC3/DSPC/Chol/PEG-
DSG/GalNAc-PEG-DSG
50/10/35/4.5/0.5
Lipid:siRNA: 11:1
LNP16
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP17
MC3
MC3/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP18
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 12:1
LNP19
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/35/5
Lipid:siRNA: 8:1
LNP20
MC3
MC3/DSPC/Chol/PEG-DPG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP22
XTC
XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
DSPC: distearoylphosphatidylcholine
DPPC: dipalmitoylphosphatidylcholine
PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference.
ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
Synthesis of Ionizable/Cationic Lipids
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN,—ORx,—NRxRy,—NRxC(═O)Ry,—NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl,—ORx, heterocycle, —NRxRy,—NRxC(═O)Ry,—NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy,—SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
Synthesis of 515
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]−232.3 (96.94%).
Synthesis of 517A and 517B
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3(1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS−[M+H]−266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR 6=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6, Found 654.6.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen
(Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
C. Additional Formulations
i. Emulsions
The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
ii. Microemulsions
In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
iii. Microparticles
an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.
iv. Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstras se, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.
Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
v. Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
vi. Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
vii. Other Components
The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a disorder of lipid metabolism. Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by ANGPTL3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
VI. Methods of the Invention
The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression of ANGPTL3 may also be assessed indirectly by measuring a decrease in biological activity of ANGPTL3, e.g., a decrease in the level of serum lipid, triglycerides, cholesterol and/or free fatty acids.
In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.
A cell suitable for treatment using the methods of the invention may be any cell that expresses an ANGPTL3gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.
ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.
The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ANGPTL3 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.
In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.
In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.
The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.
In one aspect, the present invention also provides methods for inhibiting the expression of an ANGPTL3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an ANGPTL3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in ANGPTL3 gene and/or protein expression.
The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of ANGPTL3 expression, in a therapeutically effective amount of an iRNA targeting an ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting an ANGPTL3 gene.
An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.
Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.
Subjects that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression are those having a disorder of lipid metabolism, e.g., an inherited disorder of lipid metabolism or an acquired disorder of lipid metabolism. In one embodiment, a subject having disorder of lipid metabolism has hyperlipidemia. In another embodiment, a subject having a disorder of lipid metabolism has hypertriglyceridemia. Treatment of a subject that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., a subject is having eruptive xanthomas) and prophylactic treatment (e.g., the subject is not having eruptive xanthomas or a subject may be at risk of developing eruptive xanthomas).
The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting ANGPTL3 is administered in combination with, e.g., an agent useful in treating a disorder of lipid metabolism as described elsewhere herein. For example, additional agents suitable for treating a subject that would benefit from reducton in ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, may include agents that lower one or more serum lipids. Non-limiting examples of such agents may include cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors, e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). Other agents useful in treating a disorder of lipid metabolism may include bile sequestering agents, such as cholestyramine and other resins; VLDL secretion inhibitors, such as niacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterol acyl transferase inhibitors; farnesoid X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptide; and therapeutic antibodies against ANGPTL3. The additional therapeutic agents may also include agents that raise high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, the additional therapeutic agents may also include dietary supplements, e.g., fish oil. The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.
In one embodiment, the method includes administering a composition featured herein such that expression of the target ANGPTL3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target ANGPTL3 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.
Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target ANGPTL3gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.
Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.
Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ANGPTL3 or pharmaceutical composition thereof, “effective against” a disorder of lipid metabolism indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating disorder of lipid metabolisms and the related causes.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.
Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of dsRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
For example, subjects can be administered a therapeutic amount of dsRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.
The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.
Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 days. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some embodiments, the dosing may comprise a loading phase of multiple doses on consequitive days.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Transcripts
siRNA design was carried out to identify siRNAs targeting the human ANGPTL3 transcript annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/) and a cynomolgus monkey (Macaca fascicularis; henceforth “cyno”) ANGPTL3 transcript produced via sequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was done in-house, and the mRNA sequence is shown in SEQ ID NO:9. Design used the following transcripts from the NCBI collection: Human—NM_014495.2 (SEQ ID NO:1); Mouse—NM_013913.3 (SEQ ID NO:2). All siRNA duplexes were designed that shared 100% identity with the listed human and cyno transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in NCBI Gene database.
siRNA Design, Specificity, and Efficacy Prediction
The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 977 candidate human/cyno siRNAs, and a subset of 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) were then used in a comprehensive search against the human transcriptome (defined as the set of NM_ and XM_records within the human NCBI Refseq set) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start were used to create 2 heptamers and one octomer. ‘Heptamer1’ was created by adding a 3′ A to the hexamer; ‘heptamer2’ was created by adding a 5′ A to the hexamer; octomer was created by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3×normalized octomer count)+(2×heptamer2 count)+(1×heptamer1 count)).
Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. Sorting was carried out by the specificity of the antisense strand. Duplexes were then selected from the human/cyno set with antisense oligos lacking miRNA seed matches, scores of 3 or better, less than 65% overall GC content, no GC at the first position, 4 or more Us or As in the seed region, and GC at the nineteenth position. Duplexes from the human/cyno/mouse set with antisense oligos having scores of 2 or better, less than 65% overall GC content, and no GC at the first position were also selected.
siRNA sequence selection
A total of 47 sense and 47 antisense derived siRNA oligos from the human/cyno set were synthesized and formed into duplexes. A total of 15 sense and 15 antisense derived siRNAs from the human/cyno/mouse set were synthesized and formed into duplexes.
Synthesis of ANGPTL3 Sequences
ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either a 1 or 0.2 μmol scale. Single strands were synthesized with 2′O-methyl modifications for transfection based in vitro screening. For use in free uptake screening assays, 3′ GalNAc conjugates were made with 2′F and 2′-O-methyl chemical modifications. In these designs, GalNAc moiety was placed at the 3′end of the sense strand. The antisense sequence was 23 nucleotides in length and also contained 2′F and 2′Omethyl chemical modifications with two phosphorothioate linkages at the 3′end.
On one set of 21mer single strands and duplexes, ‘endolight’ chemistry was applied as detailed below.
All pyrimidines (cytosine and uridine) in the sense strand were modified with 2′-O-Methyl nucleotides (2′O-Methyl C and 2′-O-Methyl U)
In the antisense strand, pyrimidines adjacent (towards 5′ position) to ribo A nucleoside were replaced with their corresponding 2′-O-Methyl nucleosides
A two base dTsdT extension at the 3′ end of both sense and anti sense sequences was introduced
For GalNAc conjugated 21mer sense and complementary 23mer antisense sequences, 2′F and 2′O-Methyl modified single strands were synthesized. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support for the antisense sequence at a 1 μmol scale. The sequence motif named TOFFEE was applied, in which the sense strand contained a three-nucleotide 2′F-modified motif at positions 9, 10 and 11 and in the antisense, a 2′O-Methyl-modified motif was included at positions 11, 12 and 13.
Synthesis, Cleavage and Deprotection
The synthesis of ANGPTL3 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for the sense strand and a universal CPG for the antisesense strand were used.
The synthesis of the above sequences was performed at either a 1 or 0.2 μm scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as the activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mix and the pellets were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification, Desalting and Annealing
ANGPTL3 sequences were precipitated and purified on an AKTA Purifier system using a Sephadex column. The ANGPTL3 was run at ambient temperature. Sample injection and collection was performed in 96 well plates with 1.8 mL deep wells. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ANGPTL3 sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.
Example 2. In Vitro Screening
Cell Culture and Transfections
Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×104 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.
Free Uptake Transfection
5 μl of each GalNac conjugated siRNA in PBS was combined with 4×104 freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspended in 95 μl of In Vitro Gro CP media (In Vitro Technologies-Celsis, Baltimore, Md.) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37° C. in an atmosphere of 5% CO2. siRNAs were tested at final concentrations of 500 nM, 100 nM and 10 nM for efficacy free uptake assays. For dose response screens, final siRNA concentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM.
Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)
Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl of Wash Buffer B, captured, and the supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured, and the supernatant was removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)
A master mix of 2 μl 10× Buffer, 0.8 μl 25× dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR
2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat #Hs00205581_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC50s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).
Viability Screens
Cell viability was measured on days 3 and 6 in HeLa and Hep3B cells following transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells were plated at a density of 10,000 cells per well in 96 well plates. Each siRNA was assayed in triplicate and the data averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability, and AD-1955 and mock transfected cells as negative controls. PLK1 and AD-19200 result in a dose dependent loss of viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96 well plates after 3 or 6 days and incubated at 37° C. for 2 hours. Plates were then read in a Spectrophotometer (Molecular Devices) at 560Ex/590Em. Viability was expressed as the average value of light units from three replicate transfections+/−standard deviation. Relative viability was assessed by first averaging the three replicate transfections and then normalizing Mock transfected cells. Data is expressed as % viabile cells.
TABLE 1
Abbreviations of nucleotide monomers used in nucleic acid
sequence representation.
It will be understood that these monomers, when present in an
oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.
Abbreviation
Nucleotide(s)
A
adenosine
C
cytidine
G
guanosine
T
thymidine
U
uridine
N
any nucleotide (G, A, C, T or U)
a
2′-O-methyladenosine
c
2′-O-methylcytidine
g
2′-O-methylguanosine
u
2′-O-methyluridine
dT
2′-deoxythymidine
s
phosphorothioate linkage
TABLE 2
Unmodified sense and antisense strand sequences
of ANGPTL3 dsRNAs
Sense Sequence
(SEQ ID NOS 16-77,
respectively, in order
Position in
Duplex ID
Sense Name
of appearance)
NM_014495.2
AD-45939.1
A-96225.1
UAUUUGAUCAGUCUUUUUA
281-299
AD-45858.1
A-96149.1
GAGCAACUAACUAACUUAA
478-496
AD-45869.1
A-96137.1
GGCCAAAUUAAUGACAUAU
247-265
AD-45884.1
A-96189.1
CGAAUUGAGUUGGAAGACU
1045-1063
AD-45892.1
A-96129.1
CCUCCUUCAGUUGGGACAU
198-216
AD-45899.1
A-96147.1
CACUUGAACUCAACUCAAA
401-419
AD-45915.1
A-96231.1
GUCCAUGGACAUUAAUUCA
890-908
AD-45924.1
A-96219.1
AAUCAAGAUUUGCUAUGUU
152-170
AD-45860.1
A-96181.1
CUAGAGAAGAUAUACUCCA
1000-1018
AD-45870.1
A-96153.1
CUAACUAACUUAAUUCAAA
484-502
AD-45870.2
A-96153.2
CUAACUAACUUAAUUCAAA
484-502
AD-45877.1
A-96171.1
CAUUAAUUCAACAUCGAAU
899-917
AD-45885.1
A-96205.1
CAAAAUGUUGAUCCAUCCA
1392-1410
AD-45893.1
A-96145.1
CAUAUAAACUACAAGUCAA
359-377
AD-45900.1
A-96163.1
GACCCAGCAACUCUCAAGU
839-857
AD-45925.1
A-96235.1
GGUUGGGCCUAGAGAAGAU
992-1010
AD-45861.1
A-96197.1
GUGUGGAGAAAACAACCUA
1272-1290
AD-45871.1
A-96169.1
GACAUUAAUUCAACAUCGA
897-915
AD-45878.1
A-96187.1
CAUAGUGAAGCAAUCUAAU
1017-1035
AD-45886.1
A-96127.1
CUAUGUUAGACGAUGUAAA
164-182
AD-45894.1
A-96161.1
CACAGAAAUUUCUCUAUCU
684-702
AD-45901.1
A-96179.1
GUUGGGCCUAGAGAAGAUA
993-1011
AD-45909.1
A-96213.1
GCCAAAAUCAAGAUUUGCU
147-165
AD-45934.1
A-96223.1
ACAUAUUUGAUCAGUCUUU
278-296
AD-45934.2
A-96223.2
ACAUAUUUGAUCAGUCUUU
278-296
AD-45863.1
A-96135.1
CUUAAAGACUUUGUCCAUA
220-238
AD-45872.1
A-96185.1
CCAUAGUGAAGCAAUCUAA
1016-1034
AD-45879.1
A-96203.1
CAACCAAAAUGUUGAUCCA
1388-1406
AD-45887.1
A-96143.1
CUACAUAUAAACUACAAGU
356-374
AD-45895.1
A-96177.1
GGGAGGCUUGAUGGAGAAU
970-988
AD-45902.1
A-96195.1
GGUGUUUUCUACUUGGGAU
1188-1206
AD-45910.1
A-96229.1
AAGAGCACCAAGAACUACU
711-729
AD-45935.1
A-96239.1
UGGAGAAAACAACCUAAAU
1275-1293
AD-45864.1
A-96151.1
GCAACUAACUAACUUAAUU
480-498
AD-45873.1
A-96201.1
CAACCUAAAUGGUAAAUAU
1284-1302
AD-45880.1
A-96125.1
GCUAUGUUAGACGAUGUAA
163-181
AD-45888.1
A-96159.1
CCCACAGAAAUUUCUCUAU
682-700
AD-45896.1
A-96193.1
GAUUUGGUGUUUUCUACUU
1183-1201
AD-45903.1
A-96211.1
CAGAGCCAAAAUCAAGAUU
143-161
AD-45919.1
A-96217.1
AAAUCAAGAUUUGCUAUGU
151-169
AD-45865.1
A-96167.1
CAUGGACAUUAAUUCAACA
893-911
AD-45874.1
A-96123.1
GAUUUGCUAUGUUAGACGA
158-176
AD-45881.1
A-96141.1
GAACUACAUAUAAACUACA
353-371
AD-45889.1
A-96175.1
CGAAUAGAUGGAUCACAAA
913-931
AD-45897.1
A-96209.1
CUUGUUAAAACUCUAAACU
1817-1835
AD-45904.1
A-96227.1
AUUUGAUCAGUCUUUUUAU
282-300
AD-45920.1
A-96233.1
UCCAUGGACAUUAAUUCAA
891-909
AD-45856.1
A-96117.1
CACAAUUAAGCUCCUUCUU
57-75
AD-45929.1
A-96221.1
CAACAUAUUUGAUCAGUCU
276-294
AD-45866.1
A-96183.1
CUCCAUAGUGAAGCAAUCU
1014-1032
AD-45875.1
A-96139.1
GCCAAAUUAAUGACAUAUU
248-266
AD-45882.1
A-96157.1
CAACAGCAUAGUCAAAUAA
622-640
AD-45890.1
A-96191.1
GGAAAUCACGAAACCAACU
1105-1123
AD-45898.1
A-96131.1
CAGUUGGGACAUGGUCUUA
205-223
AD-45857.1
A-96133.1
GACAUGGUCUUAAAGACUU
212-230
AD-45930.1
A-96237.1
UGUGGAGAAAACAACCUAA
1273-1291
AD-45867.1
A-96199.1
GUGGAGAAAACAACCUAAA
1274-1292
AD-45876.1
A-96155.1
CCAACAGCAUAGUCAAAUA
621-639
AD-45883.1
A-96173.1
CAACAUCGAAUAGAUGGAU
907-925
AD-45891.1
A-96207.1
GCAAAUUUAAAAGGCAAUA
1441-1459
AD-45914.1
A-96215.1
CAAAAUCAAGAUUUGCUAU
149-167
AD-15838.1
A-26242.1
AGAGCCAAAAUCAAGAUUU
144-162
Antisense Sequence
(SEQ ID NOS 78-139,
Antisense
respectively,
Position in
Duplex ID
Name
in order of appearance)
NM_014495.2
AD-45939.1
A-96226.1
UAAAAAGACUGAUCAAAUA
281-299
AD-45858.1
A-96150.1
UUAAGUUAGUUAGUUGCUC
478-496
AD-45869.1
A-96138.1
AUAUGUCAUUAAUUUGGCC
247-265
AD-45884.1
A-96190.1
AGUCUUCCAACUCAAUUCG
1045-1063
AD-45892.1
A-96130.1
AUGUCCCAACUGAAGGAGG
198-216
AD-45899.1
A-96148.1
UUUGAGUUGAGUUCAAGUG
401-419
AD-45915.1
A-96232.1
UGAAUUAAUGUCCAUGGAC
890-908
AD-45924.1
A-96220.1
AACAUAGCAAAUCUUGAUU
152-170
AD-45860.1
A-96182.1
UGGAGUAUAUCUUCUCUAG
1000-1018
AD-45870.1
A-96154.1
UUUGAAUUAAGUUAGUUAG
484-502
AD-45870.2
A-96154.2
UUUGAAUUAAGUUAGUUAG
484-502
AD-45877.1
A-96172.1
AUUCGAUGUUGAAUUAAUG
899-917
AD-45885.1
A-96206.1
UGGAUGGAUCAACAUUUUG
1392-1410
AD-45893.1
A-96146.1
UUGACUUGUAGUUUAUAUG
359-377
AD-45900.1
A-96164.1
ACUUGAGAGUUGCUGGGUC
839-857
AD-45925.1
A-96236.1
AUCUUCUCUAGGCCCAACC
992-1010
AD-45861.1
A-96198.1
UAGGUUGUUUUCUCCACAC
1272-1290
AD-45871.1
A-96170.1
UCGAUGUUGAAUUAAUGUC
897-915
AD-45878.1
A-96188.1
AUUAGAUUGCUUCACUAUG
1017-1035
AD-45886.1
A-96128.1
UUUACAUCGUCUAACAUAG
164-182
AD-45894.1
A-96162.1
AGAUAGAGAAAUUUCUGUG
684-702
AD-45901.1
A-96180.1
UAUCUUCUCUAGGCCCAAC
993-1011
AD-45909.1
A-96214.1
AGCAAAUCUUGAUUUUGGC
147-165
AD-45934.1
A-96224.1
AAAGACUGAUCAAAUAUGU
278-296
AD-45934.2
A-96224.2
AAAGACUGAUCAAAUAUGU
278-296
AD-45863.1
A-96136.1
UAUGGACAAAGUCUUUAAG
220-238
AD-45872.1
A-96186.1
UUAGAUUGCUUCACUAUGG
1016-1034
AD-45879.1
A-96204.1
UGGAUCAACAUUUUGGUUG
1388-1406
AD-45887.1
A-96144.1
ACUUGUAGUUUAUAUGUAG
356-374
AD-45895.1
A-96178.1
AUUCUCCAUCAAGCCUCCC
970-988
AD-45902.1
A-96196.1
AUCCCAAGUAGAAAACACC
1188-1206
AD-45910.1
A-96230.1
AGUAGUUCUUGGUGCUCUU
711-729
AD-45935.1
A-96240.1
AUUUAGGUUGUUUUCUCCA
1275-1293
AD-45864.1
A-96152.1
AAUUAAGUUAGUUAGUUGC
480-498
AD-45873.1
A-96202.1
AUAUUUACCAUUUAGGUUG
1284-1302
AD-45880.1
A-96126.1
UUACAUCGUCUAACAUAGC
163-181
AD-45888.1
A-96160.1
AUAGAGAAAUUUCUGUGGG
682-700
AD-45896.1
A-96194.1
AAGUAGAAAACACCAAAUC
1183-1201
AD-45903.1
A-96212.1
AAUCUUGAUUUUGGCUCUG
143-161
AD-45919.1
A-96218.1
ACAUAGCAAAUCUUGAUUU
151-169
AD-45865.1
A-96168.1
UGUUGAAUUAAUGUCCAUG
893-911
AD-45874.1
A-96124.1
UCGUCUAACAUAGCAAAUC
158-176
AD-45881.1
A-96142.1
UGUAGUUUAUAUGUAGUUC
353-371
AD-45889.1
A-96176.1
UUUGUGAUCCAUCUAUUCG
913-931
AD-45897.1
A-96210.1
AGUUUAGAGUUUUAACAAG
1817-1835
AD-45904.1
A-96228.1
AUAAAAAGACUGAUCAAAU
282-300
AD-45920.1
A-96234.1
UUGAAUUAAUGUCCAUGGA
891-909
AD-45856.1
A-96118.1
AAGAAGGAGCUUAAUUGUG
57-75
AD-45929.1
A-96222.1
AGACUGAUCAAAUAUGUUG
276-294
AD-45866.1
A-96184.1
AGAUUGCUUCACUAUGGAG
1014-1032
AD-45875.1
A-96140.1
AAUAUGUCAUUAAUUUGGC
248-266
AD-45882.1
A-96158.1
UUAUUUGACUAUGCUGUUG
622-640
AD-45890.1
A-96192.1
AGUUGGUUUCGUGAUUUCC
1105-1123
AD-45898.1
A-96132.1
UAAGACCAUGUCCCAACUG
205-223
AD-45857.1
A-96134.1
AAGUCUUUAAGACCAUGUC
212-230
AD-45930.1
A-96238.1
UUAGGUUGUUUUCUCCACA
1273-1291
AD-45867.1
A-96200.1
UUUAGGUUGUUUUCUCCAC
1274-1292
AD-45876.1
A-96156.1
UAUUUGACUAUGCUGUUGG
621-639
AD-45883.1
A-96174.1
AUCCAUCUAUUCGAUGUUG
907-925
AD-45891.1
A-96208.1
UAUUGCCUUUUAAAUUUGC
1441-1459
AD-45914.1
A-96216.1
AUAGCAAAUCUUGAUUUUG
149-167
AD-15838.1
A-26243.2
AAAUCUUGAUUUUGGCUCU
144-162
TABLE 3
Modified sense and antisense strand sequences of ANGPTL3 dsRNAs
Sense Sequence
Antisense Sequence
(SEQ ID NOS 140-201,
(SEQ ID NOS 202-263,
Sense
respectively,
Antisense
respectively,
Duplex ID
OligoName
in order of appearance)
OligoName
in order of appearance)
AD-45939.1
A-96225.1
uAuuuGAucAGucuuuuuAdTsdT
A-96226.1
uAAAAAGACUGAUcAAAuAdTsdT
AD-45858.1
A-96149.1
GAGcAAcuAAcuAAcuuAAdTsdT
A-96150.1
UuAAGUuAGUuAGUUGCUCdTsdT
AD-45869.1
A-96137.1
GGccAAAuuAAuGAcAuAudTsdT
A-96138.1
AuAUGUcAUuAAUUUGGCCdTsdT
AD-45884.1
A-96189.1
cGAAuuGAGuuGGAAGAcudTsdT
A-96190.1
AGUCUUCcAACUcAAUUCGdTsdT
AD-45892.1
A-96129.1
ccuccuucAGuuGGGAcAudTsdT
A-96130.1
AUGUCCcAACUGAAGGAGGdTsdT
AD-45899.1
A-96147.1
cAcuuGAAcucAAcucAAAdTsdT
A-96148.1
UUUGAGUUGAGUUcAAGUGdTsdT
AD-45915.1
A-96231.1
GuccAuGGAcAuuAAuucAdTsdT
A-96232.1
UGAAUuAAUGUCcAUGGACdTsdT
AD-45924.1
A-96219.1
AAucAAGAuuuGcuAuGuudTsdT
A-96220.1
AAcAuAGcAAAUCUUGAUUdTsdT
AD-45860.1
A-96181.1
cuAGAGAAGAuAuAcuccAdTsdT
A-96182.1
UGGAGuAuAUCUUCUCuAGdTsdT
AD-45870.1
A-96153.1
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.1
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45870.2
A-96153.2
cuAAcuAAcuuAAuucAAAdTsdT
A-96154.2
UUUGAAUuAAGUuAGUuAGdTsdT
AD-45877.1
A-96171.1
cAuuAAuucAAcAucGAAudTsdT
A-96172.1
AUUCGAUGUUGAAUuAAUGdTsdT
AD-45885.1
A-96205.1
cAAAAuGuuGAuccAuccAdTsdT
A-96206.1
UGGAUGGAUcAAcAUUUUGdTsdT
AD-45893.1
A-96145.1
cAuAuAAAcuAcAAGucAAdTsdT
A-96146.1
UUGACUUGuAGUUuAuAUGdTsdT
AD-45900.1
A-96163.1
GAcccAGcAAcucucAAGudTsdT
A-96164.1
ACUUGAGAGUUGCUGGGUCdTsdT
AD-45925.1
A-96235.1
GGuuGGGccuAGAGAAGAudTsdT
A-96236.1
AUCUUCUCuAGGCCcAACCdTsdT
AD-45861.1
A-96197.1
GuGuGGAGAAAAcAAccuAdTsdT
A-96198.1
uAGGUUGUUUUCUCcAcACdTsdT
AD-45871.1
A-96169.1
GAcAuuAAuucAAcAucGAdTsdT
A-96170.1
UCGAUGUUGAAUuAAUGUCdTsdT
AD-45878.1
A-96187.1
cAuAGuGAAGcAAucuAAudTsdT
A-96188.1
AUuAGAUUGCUUcACuAUGdTsdT
AD-45886.1
A-96127.1
cuAuGuuAGAcGAuGuAAAdTsdT
A-96128.1
UUuAcAUCGUCuAAcAuAGdTsdT
AD-45894.1
A-96161.1
cAcAGAAAuuucucuAucudTsdT
A-96162.1
AGAuAGAGAAAUUUCUGUGdTsdT
AD-45901.1
A-96179.1
GuuGGGccuAGAGAAGAuAdTsdT
A-96180.1
uAUCUUCUCuAGGCCcAACdTsdT
AD-45909.1
A-96213.1
GccAAAAucAAGAuuuGcudTsdT
A-96214.1
AGcAAAUCUUGAUUUUGGCdTsdT
AD-45934.1
A-96223.1
AcAuAuuuGAucAGucuuudTsdT
A-96224.1
AAAGACUGAUcAAAuAUGUdTsdT
AD-45934.2
A-96223.2
AcAuAuuuGAucAGucuuudTsdT
A-96224.2
AAAGACUGAUcAAAuAUGUdTsdT
AD-45863.1
A-96135.1
cuuAAAGAcuuuGuccAuAdTsdT
A-96136.1
uAUGGAcAAAGUCUUuAAGdTsdT
AD-45872.1
A-96185.1
ccAuAGuGAAGcAAucuAAdTsdT
A-96186.1
UuAGAUUGCUUcACuAUGGdTsdT
AD-45879.1
A-96203.1
cAAccAAAAuGuuGAuccAdTsdT
A-96204.1
UGGAUcAAcAUUUUGGUUGdTsdT
AD-45887.1
A-96143.1
cuAcAuAuAAAcuAcAAGudTsdT
A-96144.1
ACUUGuAGUUuAuAUGuAGdTsdT
AD-45895.1
A-96177.1
GGGAGGcuuGAuGGAGAAudTsdT
A-96178.1
AUUCUCcAUcAAGCCUCCCdTsdT
AD-45902.1
A-96195.1
GGuGuuuucuAcuuGGGAudTsdT
A-96196.1
AUCCcAAGuAGAAAAcACCdTsdT
AD-45910.1
A-96229.1
AAGAGcAccAAGAAcuAcudTsdT
A-96230.1
AGuAGUUCUUGGUGCUCUUdTsdT
AD-45935.1
A-96239.1
uGGAGAAAAcAAccuAAAudTsdT
A-96240.1
AUUuAGGUUGUUUUCUCcAdTsdT
AD-45864.1
A-96151.1
GcAAcuAAcuAAcuuAAuudTsdT
A-96152.1
AAUuAAGUuAGUuAGUUGCdTsdT
AD-45873.1
A-96201.1
cAAccuAAAuGGuAAAuAudTsdT
A-96202.1
AuAUUuACcAUUuAGGUUGdTsdT
AD-45880.1
A-96125.1
GcuAuGuuAGAcGAuGuAAdTsdT
A-96126.1
UuAcAUCGUCuAAcAuAGCdTsdT
AD-45888.1
A-96159.1
cccAcAGAAAuuucucuAudTsdT
A-96160.1
AuAGAGAAAUUUCUGUGGGdTsdT
AD-45896.1
A-96193.1
GAuuuGGuGuuuucuAcuudTsdT
A-96194.1
AAGuAGAAAAcACcAAAUCdTsdT
AD-45903.1
A-96211.1
cAGAGccAAAAucAAGAuudTsdT
A-96212.1
AAUCUUGAUUUUGGCUCUGdTsdT
AD-45919.1
A-96217.1
AAAucAAGAuuuGcuAuGudTsdT
A-96218.1
AcAuAGcAAAUCUUGAUUUdTsdT
AD-45865.1
A-96167.1
cAuGGAcAuuAAuucAAcAdTsdT
A-96168.1
UGUUGAAUuAAUGUCcAUGdTsdT
AD-45874.1
A-96123.1
GAuuuGcuAuGuuAGAcGAdTsdT
A-96124.1
UCGUCuAAcAuAGcAAAUCdTsdT
AD-45881.1
A-96141.1
GAAcuAcAuAuAAAcuAcAdTsdT
A-96142.1
UGuAGUUuAuAUGuAGUUCdTsdT
AD-45889.1
A-96175.1
cGAAuAGAuGGAucAcAAAdTsdT
A-96176.1
UUUGUGAUCcAUCuAUUCGdTsdT
AD-45897.1
A-96209.1
cuuGuuAAAAcucuAAAcudTsdT
A-96210.1
AGUUuAGAGUUUuAAcAAGdTsdT
AD-45904.1
A-96227.1
AuuuGAucAGucuuuuuAudTsdT
A-96228.1
AuAAAAAGACUGAUcAAAUdTsdT
AD-45920.1
A-96233.1
uccAuGGAcAuuAAuucAAdTsdT
A-96234.1
UUGAAUuAAUGUCcAUGGAdTsdT
AD-45856.1
A-96117.1
cAcAAuuAAGcuccuucuudTsdT
A-96118.1
AAGAAGGAGCUuAAUUGUGdTsdT
AD-45929.1
A-96221.1
cAAcAuAuuuGAucAGucudTsdT
A-96222.1
AGACUGAUcAAAuAUGUUGdTsdT
AD-45866.1
A-96183.1
cuccAuAGuGAAGcAAucudTsdT
A-96184.1
AGAUUGCUUcACuAUGGAGdTsdT
AD-45875.1
A-96139.1
GccAAAuuAAuGAcAuAuudTsdT
A-96140.1
AAuAUGUcAUuAAUUUGGCdTsdT
AD-45882.1
A-96157.1
cAAcAGcAuAGucAAAuAAdTsdT
A-96158.1
UuAUUUGACuAUGCUGUUGdTsdT
AD-45890.1
A-96191.1
GGAAAucAcGAAAccAAcudTsdT
A-96192.1
AGUUGGUUUCGUGAUUUCCdTsdT
AD-45898.1
A-96131.1
cAGuuGGGAcAuGGucuuAdTsdT
A-96132.1
uAAGACcAUGUCCcAACUGdTsdT
AD-45857.1
A-96133.1
GAcAuGGucuuAAAGAcuudTsdT
A-96134.1
AAGUCUUuAAGACcAUGUCdTsdT
AD-45930.1
A-96237.1
uGuGGAGAAAAcAAccuAAdTsdT
A-96238.1
UuAGGUUGUUUUCUCcAcAdTsdT
AD-45867.1
A-96199.1
GuGGAGAAAAcAAccuAAAdTsdT
A-96200.1
UUuAGGUUGUUUUCUCcACdTsdT
AD-45876.1
A-96155.1
ccAAcAGcAuAGucAAAuAdTsdT
A-96156.1
uAUUUGACuAUGCUGUUGGdTsdT
AD-45883.1
A-96173.1
cAAcAucGAAuAGAuGGAudTsdT
A-96174.1
AUCcAUCuAUUCGAUGUUGdTsdT
AD-45891.1
A-96207.1
GcAAAuuuAAAAGGcAAuAdTsdT
A-96208.1
uAUUGCCUUUuAAAUUUGCdTsdT
AD-45914.1
A-96215.1
cAAAAucAAGAuuuGcuAudTsdT
A-96216.1
AuAGcAAAUCUUGAUUUUGdTsdT
AD-15838.1
A-26242.1
AGAGccAAAAucAAGAuuudTsdT
A-26243.2
AAAUCUuGAUUUuGGCUCUdTsdT
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; s is a phosphothiorate linkage.
TABLE 4
Results of single dose screen using ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide
duplexes listed in Table 3. The sequence of AD-15838.2 is identical
to the sequence of AD-15838.1.
Delivery of siRNA duplexes was done using LNPs.
Human Hep3B
STDEV,
STDEV,
Duplex
10 nM
0.1 nM
10 nM
0.1 nM
AD-15838.2
0.09
0.66
0.008
0.030
AD-45856.1
0.32
0.91
0.026
0.032
AD-45857.1
2.46
1.07
0.140
0.044
AD-45858.1
0.10
0.74
0.010
0.070
AD-45860.1
0.02
0.47
0.002
0.097
AD-45861.1
0.03
0.68
0.004
0.062
AD-45863.1
1.42
0.95
0.145
0.126
AD-45864.1
0.02
0.17
0.002
0.045
AD-45865.1
0.32
0.93
0.022
0.062
AD-45866.1
0.10
0.92
0.010
0.041
AD-45867.1
0.04
0.61
0.000
0.048
AD-45869.1
0.45
1.08
0.028
0.081
AD-45870.1
0.01
0.10
0.003
0.010
AD-45871.1
0.05
0.57
0.006
0.071
AD-45872.1
0.07
0.71
0.007
0.034
AD-45873.1
0.02
0.23
0.001
0.011
AD-45874.1
0.08
0.75
0.013
0.049
AD-45875.1
0.13
0.82
0.017
0.040
AD-45876.1
0.03
0.54
0.000
0.013
AD-45877.1
0.06
0.47
0.002
0.025
AD-45878.1
0.02
0.44
0.002
0.031
AD-45879.1
0.03
0.35
0.003
0.023
AD-45880.1
0.49
1.00
0.039
0.088
AD-45881.1
0.20
0.90
0.019
0.095
AD-45882.1
0.20
0.95
0.012
0.086
AD-45883.1
0.16
0.98
0.011
0.058
AD-45884.1
0.09
0.94
0.003
0.044
AD-45885.1
0.22
0.91
0.020
0.145
AD-45886.1
0.04
0.40
0.008
0.080
AD-45887.1
0.03
0.35
0.002
0.057
AD-45888.1
0.05
0.80
0.006
0.042
AD-45889.1
0.31
0.91
0.013
0.052
AD-45890.1
0.06
0.90
0.001
0.047
AD-45891.1
0.06
0.82
0.007
0.034
AD-45892.1
1.01
1.09
0.033
0.211
AD-45893.1
0.04
0.58
0.002
0.046
AD-45894.1
0.04
0.59
0.003
0.024
AD-45895.1
0.84
1.00
0.047
0.047
AD-45896.1
0.84
0.98
0.032
0.095
AD-45897.1
0.36
0.61
0.032
0.053
AD-45898.1
0.98
1.09
0.021
0.117
AD-45899.1
0.04
0.59
0.005
0.095
AD-45900.1
0.06
0.80
0.005
0.091
AD-45901.1
0.33
0.94
0.025
0.096
AD-45902.1
0.24
1.03
0.010
0.079
AD-45903.1
0.74
1.02
0.003
0.092
AD-45904.1
0.39
0.87
0.010
0.010
AD-45909.1
0.04
0.73
0.008
0.013
AD-45910.1
1.08
1.01
0.037
0.089
AD-45914.1
0.52
0.99
0.018
0.071
AD-45915.1
0.06
0.48
0.004
0.046
AD-45919.1
0.67
0.98
0.048
0.064
AD-45920.1
0.61
1.00
0.031
0.038
AD-45924.1
0.09
0.67
0.005
0.012
AD-45925.1
0.13
0.90
0.008
0.100
AD-45929.1
0.02
0.42
0.001
0.083
AD-45930.1
0.05
0.63
0.005
0.052
AD-45934.1
0.04
0.41
0.001
0.062
AD-45935.1
0.08
0.76
0.006
0.058
AD-45939.1
0.23
0.82
0.030
0.028
AD-1955.1
0.93
0.93
0.068
0.073
AD-1955.1
0.94
1.01
0.028
0.113
AD-1955.1
1.00
1.02
0.032
0.065
AD-1955.1
1.15
1.06
0.053
0.019
TABLE 5
Dose response screen results for ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide
duplexes listed in Table 3. The sequence of AD-15838.2 is
identical to the sequence of AD-15838.1.
Hep3B IC50
24 hrs
120 hrs
IC50
IC50
IC50 I
IC50 II
weighted
IC50 I
IC50 II
weighted
Duplex
(nM)
(nM)
(nM)
(nM)
(nM)
(nM)
AD-15838.2
0.027
0.006
0.017
0.657
0.937
0.800
AD-45860.1
0.006
0.002
0.004
0.045
0.032
0.039
AD-45864.1
0.002
0.001
0.002
0.046
0.042
0.044
AD-45870.1
0.002
0.001
0.001
0.011
0.008
0.010
AD-45873.1
0.005
0.004
0.005
0.037
0.025
0.031
AD-45876.1
0.032
0.006
0.019
0.269
0.045
0.156
AD-45877.1
0.018
0.012
0.015
1.660
0.538
1.091
AD-45878.1
0.023
0.015
0.019
0.252
0.131
0.190
AD-45879.1
0.002
0.003
0.003
0.023
0.029
0.026
AD-45886.1
0.004
0.004
0.004
0.030
0.018
0.025
AD-45887.1
0.010
0.009
0.010
0.058
0.059
0.059
AD-45915.1
0.016
0.015
0.015
0.110
0.056
0.083
AD-45929.1
0.023
0.008
0.016
0.227
0.025
0.124
AD-45934.1
0.006
0.006
0.006
0.110
0.045
0.077
TABLE 6
Results of cell viability screens using modified ANGPTL3 dsRNA sequences
The experiments were conducted using modified oligonucleotide duplexes
listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of
AD-15838.1. Viability data is expressed as % viable relative to mock treated cells.
Ave
Ave
Ave
Ave
Ave
SD
SD
SD
SD
SD
Target
Duplex
10 nM
1 nM
500 pM
100 pM
50 pM
10 nM
1 nM
500 pM
100 pM
50 pM
HeLa day 3
ANGPTL3
AD-15838.2
37.34
58.67
70.92
89.86
94.98
9.45
12.28
15.06
22.37
18.23
ANGPTL3
AD-15838.2
29.13
48.99
63.18
79.21
94.47
1.62
5.56
4.34
11.15
11.31
ANGPTL3
AD-45860.1
67.10
75.49
77.93
86.57
90.51
6.99
12.93
6.39
6.97
3.57
ANGPTL3
AD-45864.1
99.13
96.95
86.77
89.20
84.36
7.90
7.22
12.60
4.85
6.87
ANGPTL3
AD-45870.1
82.36
97.02
95.33
95.67
92.27
8.07
5.12
7.97
7.05
10.29
ANGPTL3
AD-45873.1
67.96
90.01
90.60
94.20
103.63
11.26
22.61
15.92
22.92
16.97
ANGPTL3
AD-45876.1
64.00
76.71
80.21
81.71
91.23
6.60
13.94
10.15
10.81
13.89
ANGPTL3
AD-45877.1
79.55
77.33
79.98
91.96
93.46
1.66
9.80
8.73
16.63
11.41
ANGPTL3
AD-45878.1
81.95
78.22
78.74
87.93
85.03
15.37
22.72
22.59
30.84
40.04
ANGPTL3
AD-45878.1
66.83
70.71
82.14
82.80
83.14
17.48
6.49
6.86
19.92
21.15
ANGPTL3
AD-45879.1
37.56
45.55
59.28
76.35
78.38
3.50
7.96
19.73
34.33
33.99
ANGPTL3
AD-45886.1
72.75
57.90
64.51
81.92
82.89
14.73
12.64
11.78
25.60
23.14
ANGPTL3
AD-45887.1
38.01
53.91
59.31
76.44
85.73
0.58
10.81
6.27
11.12
10.92
ANGPTL3
AD-45915.1
48.06
52.17
67.90
95.45
100.77
8.13
15.15
29.11
32.49
38.79
ANGPTL3
AD-45929.1
29.27
44.58
52.87
76.45
88.03
4.17
9.67
14.49
31.74
28.82
ANGPTL3
AD-45934.1
68.20
64.11
76.92
79.57
92.11
15.79
11.25
19.99
26.08
26.30
(+) control
AD-19200
41.09
85.94
95.13
101.29
96.60
9.99
25.31
24.56
32.26
26.35
(+) control
AD-19200
23.99
72.76
86.51
108.10
111.13
5.35
34.52
29.24
35.99
31.88
(−) control
AD-1955
89.65
99.87
94.59
104.04
105.10
4.57
5.94
4.19
5.78
7.46
(−) control
AD-1955
104.74
99.78
105.79
109.19
108.08
10.94
7.74
11.12
7.91
10.30
(−) control
mock
100.00
6.92
(−) control
mock
100.00
9.85
(+) control
PLK
10.66
26.65
46.16
92.42
98.78
1.70
8.65
13.47
22.99
23.48
(+) control
PLK
10.74
11.41
17.33
61.02
86.59
3.39
2.61
1.49
27.42
37.31
HeLa day 6
ANGPTL3
AD-15838.2
47.94
80.97
90.44
94.37
96.10
29.05
25.12
13.62
8.88
4.72
ANGPTL3
AD-15838.2
40.32
83.80
89.88
95.94
98.27
22.47
16.51
10.03
3.83
4.19
ANGPTL3
AD-45860.1
57.38
84.84
88.90
96.74
94.03
24.55
17.35
9.67
3.17
6.58
ANGPTL3
AD-45864.1
98.65
100.87
101.13
96.86
98.24
4.35
1.91
2.22
3.41
1.80
ANGPTL3
AD-45870.1
92.69
98.71
98.49
100.07
99.28
3.94
2.67
2.36
1.19
2.65
ANGPTL3
AD-45873.1
91.78
97.38
98.81
97.57
96.22
12.47
6.26
4.08
6.22
8.64
ANGPTL3
AD-45876.1
63.54
85.68
92.13
96.48
95.97
14.74
16.50
10.03
5.81
7.51
ANGPTL3
AD-45877.1
94.17
93.21
96.39
96.70
96.98
7.12
8.00
4.58
3.05
6.15
ANGPTL3
AD-45878.1
66.46
85.75
89.73
94.60
96.59
8.20
7.41
5.27
3.21
3.91
ANGPTL3
AD-45878.1
70.80
89.30
92.54
96.60
95.09
5.18
2.13
1.61
0.50
4.15
ANGPTL3
AD-45879.1
8.29
48.25
73.54
87.47
92.19
4.66
20.05
16.04
9.06
7.90
ANGPTL3
AD-45886.1
23.69
60.65
78.49
93.41
94.15
8.19
13.90
7.15
3.35
4.06
ANGPTL3
AD-45887.1
7.24
26.03
57.68
95.99
98.80
3.07
13.10
14.94
1.40
2.54
ANGPTL3
AD-45915.1
10.38
58.38
85.69
97.24
99.76
6.83
15.66
8.39
1.33
4.15
ANGPTL3
AD-45929.1
11.73
36.67
51.90
76.71
85.08
4.80
14.19
15.34
12.37
10.60
ANGPTL3
AD-45934.1
73.57
88.48
92.94
91.50
95.97
5.36
2.96
5.50
5.44
4.39
(+) control
AD-19200
63.58
90.14
95.44
94.65
93.28
34.11
14.32
8.78
10.90
12.13
(+) control
AD-19200
16.05
78.65
85.78
93.09
96.22
9.77
15.57
19.50
13.34
10.96
(−) control
AD-1955
93.52
97.36
97.90
99.65
100.07
5.02
1.78
0.84
0.58
1.14
(−) control
AD-1955
75.39
93.61
97.79
99.60
100.96
8.37
2.50
2.27
2.68
3.16
(−) control
mock
100.00
1.32
(−) control
mock
100.00
3.35
(+) control
PLK
3.68
55.22
63.00
89.39
95.33
1.42
30.96
33.97
15.85
8.54
(+) control
PLK
2.69
3.74
9.74
67.07
82.96
0.15
0.96
3.60
22.70
19.34
Hep3B day 3
ANGPTL3
AD-15838.2
35.33
61.00
68.79
82.74
90.41
2.41
6.21
4.21
2.61
7.07
ANGPTL3
AD-15838.2
35.34
61.04
72.14
89.71
106.88
1.49
2.61
7.37
6.48
7.13
ANGPTL3
AD-45860.1
17.79
39.25
60.57
94.28
99.85
1.07
3.51
3.57
13.09
16.41
ANGPTL3
AD-45864.1
80.35
88.19
87.01
89.39
92.09
6.93
6.98
9.42
7.41
17.05
ANGPTL3
AD-45870.1
75.00
93.30
96.64
106.29
99.08
7.10
12.24
4.01
5.95
9.64
ANGPTL3
AD-45873.1
42.68
78.45
82.26
97.11
96.58
5.17
5.04
8.31
12.11
11.33
ANGPTL3
AD-45876.1
31.37
55.00
70.69
93.49
91.00
4.39
6.09
5.47
15.11
6.38
ANGPTL3
AD-45877.1
74.45
94.60
96.70
103.77
106.75
3.27
2.44
3.45
6.10
7.40
ANGPTL3
AD-45878.1
50.22
69.65
80.49
92.77
97.37
2.51
14.94
10.44
8.21
5.30
ANGPTL3
AD-45878.1
44.85
65.39
75.67
92.83
109.67
10.10
7.76
8.56
7.78
4.97
ANGPTL3
AD-45879.1
23.73
60.81
84.59
95.72
108.68
6.43
21.36
19.62
13.69
5.95
ANGPTL3
AD-45886.1
27.19
55.35
64.97
100.18
102.09
0.97
6.65
11.46
6.91
4.08
ANGPTL3
AD-45887.1
41.70
97.18
101.91
111.27
105.18
9.26
6.81
7.36
1.72
2.23
ANGPTL3
AD-45915.1
45.10
66.31
82.22
97.97
103.30
6.91
11.84
14.79
6.54
2.48
ANGPTL3
AD-45929.1
48.58
79.14
89.96
95.00
101.37
10.40
10.29
10.52
18.24
10.53
ANGPTL3
AD-45934.1
80.15
102.93
112.82
114.16
113.98
5.28
0.62
4.19
0.75
3.99
(+) control
AD-19200
14.79
55.23
72.90
89.64
94.30
2.17
5.42
7.19
10.28
16.39
(+) control
AD-19200
22.76
92.02
101.56
106.68
113.09
6.61
18.99
7.41
9.83
10.64
(−) control
AD-1955
77.77
81.25
82.23
88.21
95.02
2.83
5.40
5.08
5.42
6.63
(−) control
AD-1955
80.42
86.70
90.23
93.46
97.04
10.53
5.70
8.14
3.27
3.45
(−) control
mock
100.00
5.77
(−) control
mock
100.00
9.79
(+) control
PLK
10.91
12.89
14.31
23.87
50.93
0.17
0.87
1.64
1.13
7.80
(+) control
PLK
13.19
16.12
22.89
55.03
94.35
0.78
0.88
8.36
18.88
9.85
Hep3B day 6
ANGPTL3
AD-15838.2
78.88
89.58
93.08
91.10
100.66
11.60
9.15
12.04
10.51
5.87
ANGPTL3
AD-15838.2
81.17
85.91
87.27
103.95
103.59
7.75
3.29
8.07
7.93
9.82
ANGPTL3
AD-45860.1
84.11
87.77
93.22
99.15
96.75
14.22
13.36
20.98
13.15
17.62
ANGPTL3
AD-45864.1
99.27
111.82
106.28
99.15
97.55
7.77
16.31
14.24
15.40
9.18
ANGPTL3
AD-45870.1
95.49
109.60
104.16
104.65
106.76
11.92
12.98
9.25
10.29
19.12
ANGPTL3
AD-45873.1
71.45
90.62
93.44
102.07
107.72
4.71
4.40
15.02
11.96
10.16
ANGPTL3
AD-45876.1
76.92
82.09
89.44
95.27
105.41
9.39
13.55
7.93
9.77
10.42
ANGPTL3
AD-45877.1
82.98
98.05
95.07
103.55
104.14
11.22
13.45
1.27
8.88
6.49
ANGPTL3
AD-45878.1
75.14
82.48
89.68
92.71
95.72
8.65
10.07
10.77
12.44
15.04
ANGPTL3
AD-45878.1
65.90
77.37
78.33
84.54
99.49
10.21
13.22
9.95
11.65
11.17
ANGPTL3
AD-45879.1
86.42
89.45
101.50
97.30
100.66
10.59
10.12
19.77
13.19
9.54
ANGPTL3
AD-45886.1
91.15
79.31
80.76
86.52
94.04
12.89
11.88
5.38
4.92
6.80
ANGPTL3
AD-45887.1
91.67
103.38
107.88
100.05
102.05
10.80
14.84
19.18
13.72
18.00
ANGPTL3
AD-45915.1
81.97
85.91
91.81
94.95
102.13
18.49
19.30
7.19
12.72
16.64
ANGPTL3
AD-45929.1
61.92
79.39
87.28
88.09
96.00
6.80
10.76
5.80
10.68
16.66
ANGPTL3
AD-45934.1
85.84
89.66
97.67
99.91
102.54
12.39
14.25
4.74
9.51
4.28
(+) control
AD-19200
50.48
65.62
79.67
98.61
96.87
4.60
4.64
7.20
5.08
7.37
(+) control
AD-19200
52.01
75.89
92.59
101.47
99.66
4.35
20.87
13.57
6.50
11.76
(−) control
AD-1955
91.77
95.87
93.06
95.10
97.52
8.87
3.46
1.46
2.00
3.84
(−) control
AD-1955
93.65
94.41
89.42
100.59
103.91
9.91
14.90
6.80
11.99
10.31
(−) control
mock
100.00
5.10
(−) control
mock
100.00
7.35
(+) control
PLK
36.43
37.75
40.19
55.25
64.59
3.44
2.75
3.65
5.33
5.02
(+) control
PLK
38.70
43.68
50.32
75.17
89.62
3.40
3.85
8.10
10.54
10.69
TABLE 7
Unmodified sense and antisense strand sequences of
ANGPTL3 GalNac-conjugated dsRNAs
Sense Sequence
(SEQ ID NOS 264-448,
Sense
respectively, in order
Position in
Duplex ID
Name
of appearance)
NM_014495.2
AD-53063.1
A-108558.1
AAAGACAACAAACAUUAUAUUx
1066-1086
AD-52965.1
A-108310.1
ACAAUUAAGCUCCUUCUUUUUx
58-78
AD-53030.1
A-108410.1
UGUCACUUGAACUCAACUCAAx
398-418
AD-52953.1
A-108306.1
UCACAAUUAAGCUCCUUCUUUx
56-76
AD-53001.1
A-108416.1
CUUGAACUCAACUCAAAACUUx
403-423
AD-53080.1
A-108548.1
CUCCAUAGUGAAGCAAUCUAAx
1014-1034
AD-52971.1
A-108312.1
CAAUUAAGCUCCUUCUUUUUAx
59-79
AD-53071.1
A-108498.1
ACCCAGCAACUCUCAAGUUUUx
840-860
AD-53024.1
A-108408.1
GAAUAUGUCACUUGAACUCAAx
393-413
AD-52977.1
A-108314.1
AAUUAAGCUCCUUCUUUUUAUx
60-80
AD-53064.1
A-108574.1
CAUUAUAUUGAAUAUUCUUUUx
1078-1098
AD-53033.1
A-108458.1
ACUAACUAACUUAAUUCAAAAx
483-503
AD-52954.1
A-108322.1
UUAUUGUUCCUCUAGUUAUUUx
77-97
AD-53098.1
A-108554.1
CAUAGUGAAGCAAUCUAAUUAx
1017-1037
AD-53092.1
A-108552.1
CCAUAGUGAAGCAAUCUAAUUx
1016-1036
AD-53073.1
A-108530.1
GAUCACAAAACUUCAAUGAAAx
923-943
AD-53132.1
A-108628.1
AUGGAAGGUUAUACUCUAUAAx
1364-1384
AD-53086.1
A-108550.1
UCCAUAGUGAAGCAAUCUAAUx
1015-1035
AD-52961.1
A-108340.1
CUAUGUUAGACGAUGUAAAAAx
164-184
AD-52983.1
A-108316.1
AUUAAGCUCCUUCUUUUUAUUx
61-81
AD-53027.1
A-108456.1
AACUAACUAACUUAAUUCAAAx
482-502
AD-52986.1
A-108364.1
GGCCAAAUUAAUGACAUAUUUx
247-267
AD-52989.1
A-108318.1
UUUUAUUGUUCCUCUAGUUAUx
75-95
AD-52981.1
A-108378.1
ACAUAUUUGAUCAGUCUUUUUx
278-298
AD-53077.1
A-108500.1
CCCAGCAACUCUCAAGUUUUUx
841-861
AD-53095.1
A-108506.1
CAGGUAGUCCAUGGACAUUAAx
884-904
AD-52970.1
A-108390.1
ACUGAGAAGAACUACAUAUAAx
345-365
AD-53015.1
A-108452.1
GAGCAACUAACUAACUUAAUUx
478-498
AD-53147.1
A-108618.1
AACAACCUAAAUGGUAAAUAUx
1282-1302
AD-53103.1
A-108540.1
CCUAGAGAAGAUAUACUCCAUx
999-1019
AD-52969.1
A-108374.1
CAACAUAUUUGAUCAGUCUUUx
276-296
AD-53075.1
A-108562.1
ACAACAAACAUUAUAUUGAAUx
1070-1090
AD-52994.1
A-108398.1
ACAUAUAAACUACAAGUCAAAx
358-378
AD-52960.1
A-108324.1
CUAGUUAUUUCCUCCAGAAUUx
88-108
AD-53003.1
A-108448.1
AAGAGCAACUAACUAACUUAAx
476-496
AD-52995.1
A-108320.1
UUUAUUGUUCCUCUAGUUAUUx
76-96
AD-53037.1
A-108428.1
CUCCUAGAAGAAAAAAUUCUAx
430-450
AD-53087.1
A-108566.1
AACAAACAUUAUAUUGAAUAUx
1072-1092
AD-53076.1
A-108578.1
GGAAAUCACGAAACCAACUAUx
1105-1125
AD-52975.1
A-108376.1
AACAUAUUUGAUCAGUCUUUUx
277-297
AD-53138.1
A-108630.1
UGGAAGGUUAUACUCUAUAAAx
1365-1385
AD-53091.1
A-108536.1
GGAGAACUACAAAUAUGGUUUx
948-968
AD-53124.1
A-108594.1
GAAAACAAAGAUUUGGUGUUUx
1174-1194
AD-53125.1
A-108610.1
AGUGUGGAGAAAACAACCUAAx
1271-1291
AD-53036.1
A-108412.1
GUCACUUGAACUCAACUCAAAx
399-419
AD-53061.1
A-108526.1
GAUGGAUCACAAAACUUCAAUx
919-939
AD-53093.1
A-108568.1
ACAAACAUUAUAUUGAAUAUUx
1073-1093
AD-53137.1
A-108614.1
UGUGGAGAAAACAACCUAAAUx
1273-1293
AD-52999.1
A-108384.1
AUCAGUCUUUUUAUGAUCUAUx
287-307
AD-53069.1
A-108560.1
GACAACAAACAUUAUAUUGAAx
1069-1089
AD-53034.1
A-108474.1
CAACAGCAUAGUCAAAUAAAAx
622-642
AD-52976.1
A-108392.1
CUGAGAAGAACUACAUAUAAAx
346-366
AD-52996.1
A-108336.1
UGCUAUGUUAGACGAUGUAAAx
162-182
AD-53029.1
A-108488.1
AACCCACAGAAAUUUCUCUAUx
680-700
AD-53020.1
A-108438.1
CUUCAACAAAAAGUGAAAUAUx
451-471
AD-53042.1
A-108414.1
UCACUUGAACUCAACUCAAAAx
400-420
AD-53011.1
A-108482.1
CAUAGUCAAAUAAAAGAAAUAx
628-648
AD-52957.1
A-108370.1
CAAAAACUCAACAUAUUUGAUx
268-288
AD-53008.1
A-108434.1
UACUUCAACAAAAAGUGAAAUx
449-469
AD-53065.1
A-108496.1
GACCCAGCAACUCUCAAGUUUx
839-859
AD-53115.1
A-108638.1
UUGAAUGAACUGAGGCAAAUUx
1427-1447
AD-53012.1
A-108404.1
UAUAAACUACAAGUCAAAAAUx
361-381
AD-53004.1
A-108464.1
AAACAAGAUAAUAGCAUCAAAx
559-579
AD-53021.1
A-108454.1
CAACUAACUAACUUAAUUCAAx
481-501
AD-52955.1
A-108338.1
GCUAUGUUAGACGAUGUAAAAx
163-183
AD-53119.1
A-108608.1
ACUUGGGAUCACAAAGCAAAAx
1198-1218
AD-52990.1
A-108334.1
UUGCUAUGUUAGACGAUGUAAx
161-181
AD-52964.1
A-108388.1
AACUGAGAAGAACUACAUAUAx
344-364
AD-52973.1
A-108344.1
GAUGUAAAAAUUUUAGCCAAUx
175-195
AD-53074.1
A-108546.1
ACUCCAUAGUGAAGCAAUCUAx
1013-1033
AD-53026.1
A-108440.1
UUCAACAAAAAGUGAAAUAUUx
452-472
AD-53062.1
A-108542.1
CUAGAGAAGAUAUACUCCAUAx
1000-1020
AD-53114.1
A-108622.1
CAACCUAAAUGGUAAAUAUAAx
1284-1304
AD-53082.1
A-108580.1
GAAAUCACGAAACCAACUAUAx
1106-1126
AD-53035.1
A-108490.1
CCACAGAAAUUUCUCUAUCUUx
683-703
AD-52978.1
A-108330.1
AAAUCAAGAUUUGCUAUGUUAx
151-171
AD-53084.1
A-108518.1
ACAUUAAUUCAACAUCGAAUAx
898-918
AD-52972.1
A-108328.1
CCAGAGCCAAAAUCAAGAUUUx
142-162
AD-53002.1
A-108432.1
CUACUUCAACAAAAAGUGAAAx
448-468
AD-53078.1
A-108516.1
GACAUUAAUUCAACAUCGAAUx
897-917
AD-53072.1
A-108514.1
GGACAUUAAUUCAACAUCGAAx
896-916
AD-53005.1
A-108480.1
GCAUAGUCAAAUAAAAGAAAUx
627-647
AD-53083.1
A-108502.1
CUCUCAAGUUUUUCAUGUCUAx
849-869
AD-53102.1
A-108524.1
AUCGAAUAGAUGGAUCACAAAx
911-931
AD-53105.1
A-108572.1
ACAUUAUAUUGAAUAUUCUUUx
1077-1097
AD-53090.1
A-108520.1
UUAAUUCAACAUCGAAUAGAUx
901-921
AD-53010.1
A-108466.1
GAUAAUAGCAUCAAAGACCUUx
565-585
AD-52998.1
A-108368.1
UGACAUAUUUCAAAAACUCAAx
258-278
AD-52992.1
A-108366.1
AAAUUAAUGACAUAUUUCAAAx
251-271
AD-53068.1
A-108544.1
GAAGAUAUACUCCAUAGUGAAx
1005-1025
AD-53032.1
A-108442.1
AAUAUUUAGAAGAGCAACUAAx
467-487
AD-52967.1
A-108342.1
CGAUGUAAAAAUUUUAGCCAAx
174-194
AD-53096.1
A-108522.1
UUCAACAUCGAAUAGAUGGAUx
905-925
AD-53131.1
A-108612.1
GUGUGGAGAAAACAACCUAAAx
1272-1292
AD-52963.1
A-108372.1
UCAACAUAUUUGAUCAGUCUUx
275-295
AD-53089.1
A-108504.1
UCAGGUAGUCCAUGGACAUUAx
883-903
AD-53044.1
A-108446.1
UUUAGAAGAGCAACUAACUAAx
471-491
AD-52988.1
A-108396.1
UACAUAUAAACUACAAGUCAAx
357-377
AD-53067.1
A-108528.1
GGAUCACAAAACUUCAAUGAAx
922-942
AD-53009.1
A-108450.1
AGAGCAACUAACUAACUUAAUx
477-497
AD-53022.1
A-108470.1
ACCAACAGCAUAGUCAAAUAAx
620-640
AD-53016.1
A-108468.1
AACCAACAGCAUAGUCAAAUAx
619-639
AD-53007.1
A-108418.1
GAACUCAACUCAAAACUUGAAx
406-426
AD-53148.1
A-108634.1
UACUCUAUAAAAUCAACCAAAx
1375-1395
AD-53040.1
A-108476.1
CAGCAUAGUCAAAUAAAAGAAx
625-645
AD-53041.1
A-108492.1
GAAAUAAGAAAUGUAAAACAUx
748-768
AD-53039.1
A-108460.1
CUAACUAACUUAAUUCAAAAUx
484-504
AD-53139.1
A-108646.1
AUGAACUGAGGCAAAUUUAAAx
1431-1451
AD-53144.1
A-108648.1
UGAACUGAGGCAAAUUUAAAAx
1432-1452
AD-53142.1
A-108616.1
AAACAACCUAAAUGGUAAAUAx
1281-1301
AD-53108.1
A-108620.1
ACAACCUAAAUGGUAAAUAUAx
1283-1303
AD-53079.1
A-108532.1
AACGUGGGAGAACUACAAAUAx
942-962
AD-53133.1
A-108644.1
AAUGAACUGAGGCAAAUUUAAx
1430-1450
AD-53104.1
A-108556.1
GUUGGAAGACUGGAAAGACAAx
1053-1073
AD-53088.1
A-108582.1
UGGCAAUGUCCCCAAUGCAAUx
1149-1169
AD-53101.1
A-108508.1
GGUAGUCCAUGGACAUUAAUUx
886-906
AD-53000.1
A-108400.1
CAUAUAAACUACAAGUCAAAAx
359-379
AD-53112.1
A-108590.1
AAUCCCGGAAAACAAAGAUUUx
1167-1187
AD-53107.1
A-108604.1
CUACUUGGGAUCACAAAGCAAx
1196-1216
AD-53121.1
A-108640.1
UGAAUGAACUGAGGCAAAUUUx
1428-1448
AD-53046.1
A-108478.1
AGCAUAGUCAAAUAAAAGAAAx
626-646
AD-53038.1
A-108444.1
AUUUAGAAGAGCAACUAACUAx
470-490
AD-53140.1
A-108662.1
AGGCAAAUUUAAAAGGCAAUAx
1439-1459
AD-52987.1
A-108380.1
CAUAUUUGAUCAGUCUUUUUAx
279-299
AD-53130.1
A-108596.1
AAAACAAAGAUUUGGUGUUUUx
1175-1195
AD-53106.1
A-108588.1
CAAUCCCGGAAAACAAAGAUUx
1166-1186
AD-53081.1
A-108564.1
CAACAAACAUUAUAUUGAAUAx
1071-1091
AD-53118.1
A-108592.1
GGAAAACAAAGAUUUGGUGUUx
1173-1193
AD-53136.1
A-108598.1
ACAAAGAUUUGGUGUUUUCUAx
1178-1198
AD-53127.1
A-108642.1
GAAUGAACUGAGGCAAAUUUAx
1429-1449
AD-53066.1
A-108512.1
CCAUGGACAUUAAUUCAACAUx
892-912
AD-53013.1
A-108420.1
AACUCAACUCAAAACUUGAAAx
407-427
AD-52991.1
A-108350.1
CAGUUGGGACAUGGUCUUAAAx
205-225
AD-53099.1
A-108570.1
AACAUUAUAUUGAAUAUUCUUx
1076-1096
AD-52958.1
A-108386.1
ACCAGUGAAAUCAAAGAAGAAx
316-336
AD-53097.1
A-108538.1
GUUGGGCCUAGAGAAGAUAUAx
993-1013
AD-52966.1
A-108326.1
CUCCAGAGCCAAAAUCAAGAUx
140-160
AD-53145.1
A-108664.1
GGCAAAUUUAAAAGGCAAUAAx
1440-1460
AD-53113.1
A-108606.1
UACUUGGGAUCACAAAGCAAAx
1197-1217
AD-52993.1
A-108382.1
GAUCAGUCUUUUUAUGAUCUAx
286-306
AD-53031.1
A-108426.1
GAAAGCCUCCUAGAAGAAAAAx
424-444
AD-53017.1
A-108484.1
AGUCAAAUAAAAGAAAUAGAAx
631-651
AD-53143.1
A-108632.1
AUACUCUAUAAAAUCAACCAAx
1374-1394
AD-53149.1
A-108650.1
GAACUGAGGCAAAUUUAAAAAx
1433-1453
AD-53059.1
A-108494.1
AGACCCAGCAACUCUCAAGUUx
838-858
AD-53006.1
A-108402.1
AUAUAAACUACAAGUCAAAAAx
360-380
AD-53025.1
A-108424.1
UGAAAGCCUCCUAGAAGAAAAx
423-443
AD-53085.1
A-108534.1
GGGAGAACUACAAAUAUGGUUx
947-967
AD-52984.1
A-108332.1
AGAUUUGCUAUGUUAGACGAUx
157-177
AD-53023.1
A-108486.1
GAACCCACAGAAAUUUCUCUAx
679-699
AD-53014.1
A-108436.1
ACUUCAACAAAAAGUGAAAUAx
450-470
AD-53060.1
A-108510.1
AGUCCAUGGACAUUAAUUCAAx
889-909
AD-53110.1
A-108652.1
AACUGAGGCAAAUUUAAAAGAx
1434-1454
AD-52980.1
A-108362.1
GGGCCAAAUUAAUGACAUAUUx
246-266
AD-53109.1
A-108636.1
AUCCAUCCAACAGAUUCAGAAx
1402-1422
AD-53141.1
A-108600.1
AAGAUUUGGUGUUUUCUACUUx
1181-1201
AD-53126.1
A-108626.1
GUCUCAAAAUGGAAGGUUAUAx
1356-1376
AD-53116.1
A-108654.1
ACUGAGGCAAAUUUAAAAGGAx
1435-1455
AD-52997.1
A-108352.1
GGGACAUGGUCUUAAAGACUUx
210-230
AD-53120.1
A-108624.1
AUGGUAAAUAUAACAAACCAAx
1292-1312
AD-53070.1
A-108576.1
GGGAAAUCACGAAACCAACUAx
1104-1124
AD-53028.1
A-108472.1
CCAACAGCAUAGUCAAAUAAAx
621-641
AD-53146.1
A-108602.1
UUUUCUACUUGGGAUCACAAAx
1192-1212
AD-52982.1
A-108394.1
AGAACUACAUAUAAACUACAAx
352-372
AD-53111.1
A-108668.1
AGAGUAUGUGUAAAAAUCUGUx
1915-1935
AD-53045.1
A-108462.1
AAAACAAGAUAAUAGCAUCAAx
558-578
AD-53123.1
A-108672.1
AGUAUGUGUAAAAAUCUGUAAx
1917-1937
AD-53018.1
A-108406.1
AGUCAAAAAUGAAGAGGUAAAx
372-392
AD-52956.1
A-108354.1
GGACAUGGUCUUAAAGACUUUx
211-231
AD-53134.1
A-108660.1
GAGGCAAAUUUAAAAGGCAAUx
1438-1458
AD-52968.1
A-108358.1
GUCUUAAAGACUUUGUCCAUAx
218-238
AD-53122.1
A-108656.1
CUGAGGCAAAUUUAAAAGGCAx
1436-1456
AD-53100.1
A-108586.1
GCAAUCCCGGAAAACAAAGAUx
1165-1185
AD-53128.1
A-108658.1
UGAGGCAAAUUUAAAAGGCAAx
1437-1457
AD-53043.1
A-108430.1
UCUACUUCAACAAAAAGUGAAx
447-467
AD-53135.1
A-108676.1
UAUGUGUAAAAAUCUGUAAUAx
1919-1939
AD-53094.1
A-108584.1
AAUGCAAUCCCGGAAAACAAAx
1162-1182
AD-53019.1
A-108422.1
CUUGAAAGCCUCCUAGAAGAAx
421-441
AD-53129.1
A-108674.1
GUAUGUGUAAAAAUCUGUAAUx
1918-1938
AD-53150.1
A-108666.1
CAGAGUAUGUGUAAAAAUCUUx
1914-1934
AD-53117.1
A-108670.1
GAGUAUGUGUAAAAAUCUGUAx
1916-1936
AD-52985.1
A-108348.1
UCAGUUGGGACAUGGUCUUAAx
204-224
AD-52962.1
A-108356.1
GGUCUUAAAGACUUUGUCCAUx
217-237
AD-52974.1
A-108360.1
UCUUAAAGACUUUGUCCAUAAx
219-239
AD-52979.1
A-108346.1
UUCAGUUGGGACAUGGUCUUAx
203-223
Antisense Sequence
(SEQ ID NOS 449-633,
Antisense
respectively,
Position in
Duplex ID
Name
in order of appearance)
NM_014495.2
AD-53063.1
A-108559.1
AAUAUAAUGUUUGUUGUCUUUCC
1064-1086
AD-52965.1
A-108311.1
AAAAAGAAGGAGCUUAAUUGUGA
56-78
AD-53030.1
A-108411.1
UUGAGUUGAGUUCAAGUGACAUA
396-418
AD-52953.1
A-108307.1
AAAGAAGGAGCUUAAUUGUGAAC
54-76
AD-53001.1
A-108417.1
AAGUUUUGAGUUGAGUUCAAGUG
401-423
AD-53080.1
A-108549.1
UUAGAUUGCUUCACUAUGGAGUA
1012-1034
AD-52971.1
A-108313.1
UAAAAAGAAGGAGCUUAAUUGUG
57-79
AD-53071.1
A-108499.1
AAAACUUGAGAGUUGCUGGGUCU
838-860
AD-53024.1
A-108409.1
UUGAGUUCAAGUGACAUAUUCUU
391-413
AD-52977.1
A-108315.1
AUAAAAAGAAGGAGCUUAAUUGU
58-80
AD-53064.1
A-108575.1
AAAAGAAUAUUCAAUAUAAUGUU
1076-1098
AD-53033.1
A-108459.1
UUUUGAAUUAAGUUAGUUAGUUG
481-503
AD-52954.1
A-108323.1
AAAUAACUAGAGGAACAAUAAAA
75-97
AD-53098.1
A-108555.1
UAAUUAGAUUGCUUCACUAUGGA
1015-1037
AD-53092.1
A-108553.1
AAUUAGAUUGCUUCACUAUGGAG
1014-1036
AD-53073.1
A-108531.1
UUUCAUUGAAGUUUUGUGAUCCA
921-943
AD-53132.1
A-108629.1
UUAUAGAGUAUAACCUUCCAUUU
1362-1384
AD-53086.1
A-108551.1
AUUAGAUUGCUUCACUAUGGAGU
1013-1035
AD-52961.1
A-108341.1
UUUUUACAUCGUCUAACAUAGCA
162-184
AD-52983.1
A-108317.1
AAUAAAAAGAAGGAGCUUAAUUG
59-81
AD-53027.1
A-108457.1
UUUGAAUUAAGUUAGUUAGUUGC
480-502
AD-52986.1
A-108365.1
AAAUAUGUCAUUAAUUUGGCCCU
245-267
AD-52989.1
A-108319.1
AUAACUAGAGGAACAAUAAAAAG
73-95
AD-52981.1
A-108379.1
AAAAAGACUGAUCAAAUAUGUUG
276-298
AD-53077.1
A-108501.1
AAAAACUUGAGAGUUGCUGGGUC
839-861
AD-53095.1
A-108507.1
UUAAUGUCCAUGGACUACCUGAU
882-904
AD-52970.1
A-108391.1
UUAUAUGUAGUUCUUCUCAGUUC
343-365
AD-53015.1
A-108453.1
AAUUAAGUUAGUUAGUUGCUCUU
476-498
AD-53147.1
A-108619.1
AUAUUUACCAUUUAGGUUGUUUU
1280-1302
AD-53103.1
A-108541.1
AUGGAGUAUAUCUUCUCUAGGCC
997-1019
AD-52969.1
A-108375.1
AAAGACUGAUCAAAUAUGUUGAG
274-296
AD-53075.1
A-108563.1
AUUCAAUAUAAUGUUUGUUGUCU
1068-1090
AD-52994.1
A-108399.1
UUUGACUUGUAGUUUAUAUGUAG
356-378
AD-52960.1
A-108325.1
AAUUCUGGAGGAAAUAACUAGAG
86-108
AD-53003.1
A-108449.1
UUAAGUUAGUUAGUUGCUCUUCU
474-496
AD-52995.1
A-108321.1
AAUAACUAGAGGAACAAUAAAAA
74-96
AD-53037.1
A-108429.1
UAGAAUUUUUUCUUCUAGGAGGC
428-450
AD-53087.1
A-108567.1
AUAUUCAAUAUAAUGUUUGUUGU
1070-1092
AD-53076.1
A-108579.1
AUAGUUGGUUUCGUGAUUUCCCA
1103-1125
AD-52975.1
A-108377.1
AAAAGACUGAUCAAAUAUGUUGA
275-297
AD-53138.1
A-108631.1
UUUAUAGAGUAUAACCUUCCAUU
1363-1385
AD-53091.1
A-108537.1
AAACCAUAUUUGUAGUUCUCCCA
946-968
AD-53124.1
A-108595.1
AAACACCAAAUCUUUGUUUUCCG
1172-1194
AD-53125.1
A-108611.1
UUAGGUUGUUUUCUCCACACUCA
1269-1291
AD-53036.1
A-108413.1
UUUGAGUUGAGUUCAAGUGACAU
397-419
AD-53061.1
A-108527.1
AUUGAAGUUUUGUGAUCCAUCUA
917-939
AD-53093.1
A-108569.1
AAUAUUCAAUAUAAUGUUUGUUG
1071-1093
AD-53137.1
A-108615.1
AUUUAGGUUGUUUUCUCCACACU
1271-1293
AD-52999.1
A-108385.1
AUAGAUCAUAAAAAGACUGAUCA
285-307
AD-53069.1
A-108561.1
UUCAAUAUAAUGUUUGUUGUCUU
1067-1089
AD-53034.1
A-108475.1
UUUUAUUUGACUAUGCUGUUGGU
620-642
AD-52976.1
A-108393.1
UUUAUAUGUAGUUCUUCUCAGUU
344-366
AD-52996.1
A-108337.1
UUUACAUCGUCUAACAUAGCAAA
160-182
AD-53029.1
A-108489.1
AUAGAGAAAUUUCUGUGGGUUCU
678-700
AD-53020.1
A-108439.1
AUAUUUCACUUUUUGUUGAAGUA
449-471
AD-53042.1
A-108415.1
UUUUGAGUUGAGUUCAAGUGACA
398-420
AD-53011.1
A-108483.1
UAUUUCUUUUAUUUGACUAUGCU
626-648
AD-52957.1
A-108371.1
AUCAAAUAUGUUGAGUUUUUGAA
266-288
AD-53008.1
A-108435.1
AUUUCACUUUUUGUUGAAGUAGA
447-469
AD-53065.1
A-108497.1
AAACUUGAGAGUUGCUGGGUCUG
837-859
AD-53115.1
A-108639.1
AAUUUGCCUCAGUUCAUUCAAAG
1425-1447
AD-53012.1
A-108405.1
AUUUUUGACUUGUAGUUUAUAUG
359-381
AD-53004.1
A-108465.1
UUUGAUGCUAUUAUCUUGUUUUU
557-579
AD-53021.1
A-108455.1
UUGAAUUAAGUUAGUUAGUUGCU
479-501
AD-52955.1
A-108339.1
UUUUACAUCGUCUAACAUAGCAA
161-183
AD-53119.1
A-108609.1
UUUUGCUUUGUGAUCCCAAGUAG
1196-1218
AD-52990.1
A-108335.1
UUACAUCGUCUAACAUAGCAAAU
159-181
AD-52964.1
A-108389.1
UAUAUGUAGUUCUUCUCAGUUCC
342-364
AD-52973.1
A-108345.1
AUUGGCUAAAAUUUUUACAUCGU
173-195
AD-53074.1
A-108547.1
UAGAUUGCUUCACUAUGGAGUAU
1011-1033
AD-53026.1
A-108441.1
AAUAUUUCACUUUUUGUUGAAGU
450-472
AD-53062.1
A-108543.1
UAUGGAGUAUAUCUUCUCUAGGC
998-1020
AD-53114.1
A-108623.1
UUAUAUUUACCAUUUAGGUUGUU
1282-1304
AD-53082.1
A-108581.1
UAUAGUUGGUUUCGUGAUUUCCC
1104-1126
AD-53035.1
A-108491.1
AAGAUAGAGAAAUUUCUGUGGGU
681-703
AD-52978.1
A-108331.1
UAACAUAGCAAAUCUUGAUUUUG
149-171
AD-53084.1
A-108519.1
UAUUCGAUGUUGAAUUAAUGUCC
896-918
AD-52972.1
A-108329.1
AAAUCUUGAUUUUGGCUCUGGAG
140-162
AD-53002.1
A-108433.1
UUUCACUUUUUGUUGAAGUAGAA
446-468
AD-53078.1
A-108517.1
AUUCGAUGUUGAAUUAAUGUCCA
895-917
AD-53072.1
A-108515.1
UUCGAUGUUGAAUUAAUGUCCAU
894-916
AD-53005.1
A-108481.1
AUUUCUUUUAUUUGACUAUGCUG
625-647
AD-53083.1
A-108503.1
UAGACAUGAAAAACUUGAGAGUU
847-869
AD-53102.1
A-108525.1
UUUGUGAUCCAUCUAUUCGAUGU
909-931
AD-53105.1
A-108573.1
AAAGAAUAUUCAAUAUAAUGUUU
1075-1097
AD-53090.1
A-108521.1
AUCUAUUCGAUGUUGAAUUAAUG
899-921
AD-53010.1
A-108467.1
AAGGUCUUUGAUGCUAUUAUCUU
563-585
AD-52998.1
A-108369.1
UUGAGUUUUUGAAAUAUGUCAUU
256-278
AD-52992.1
A-108367.1
UUUGAAAUAUGUCAUUAAUUUGG
249-271
AD-53068.1
A-108545.1
UUCACUAUGGAGUAUAUCUUCUC
1003-1025
AD-53032.1
A-108443.1
UUAGUUGCUCUUCUAAAUAUUUC
465-487
AD-52967.1
A-108343.1
UUGGCUAAAAUUUUUACAUCGUC
172-194
AD-53096.1
A-108523.1
AUCCAUCUAUUCGAUGUUGAAUU
903-925
AD-53131.1
A-108613.1
UUUAGGUUGUUUUCUCCACACUC
1270-1292
AD-52963.1
A-108373.1
AAGACUGAUCAAAUAUGUUGAGU
273-295
AD-53089.1
A-108505.1
UAAUGUCCAUGGACUACCUGAUA
881-903
AD-53044.1
A-108447.1
UUAGUUAGUUGCUCUUCUAAAUA
469-491
AD-52988.1
A-108397.1
UUGACUUGUAGUUUAUAUGUAGU
355-377
AD-53067.1
A-108529.1
UUCAUUGAAGUUUUGUGAUCCAU
920-942
AD-53009.1
A-108451.1
AUUAAGUUAGUUAGUUGCUCUUC
475-497
AD-53022.1
A-108471.1
UUAUUUGACUAUGCUGUUGGUUU
618-640
AD-53016.1
A-108469.1
UAUUUGACUAUGCUGUUGGUUUA
617-639
AD-53007.1
A-108419.1
UUCAAGUUUUGAGUUGAGUUCAA
404-426
AD-53148.1
A-108635.1
UUUGGUUGAUUUUAUAGAGUAUA
1373-1395
AD-53040.1
A-108477.1
UUCUUUUAUUUGACUAUGCUGUU
623-645
AD-53041.1
A-108493.1
AUGUUUUACAUUUCUUAUUUCAU
746-768
AD-53039.1
A-108461.1
AUUUUGAAUUAAGUUAGUUAGUU
482-504
AD-53139.1
A-108647.1
UUUAAAUUUGCCUCAGUUCAUUC
1429-1451
AD-53144.1
A-108649.1
UUUUAAAUUUGCCUCAGUUCAUU
1430-1452
AD-53142.1
A-108617.1
UAUUUACCAUUUAGGUUGUUUUC
1279-1301
AD-53108.1
A-108621.1
UAUAUUUACCAUUUAGGUUGUUU
1281-1303
AD-53079.1
A-108533.1
UAUUUGUAGUUCUCCCACGUUUC
940-962
AD-53133.1
A-108645.1
UUAAAUUUGCCUCAGUUCAUUCA
1428-1450
AD-53104.1
A-108557.1
UUGUCUUUCCAGUCUUCCAACUC
1051-1073
AD-53088.1
A-108583.1
AUUGCAUUGGGGACAUUGCCAGU
1147-1169
AD-53101.1
A-108509.1
AAUUAAUGUCCAUGGACUACCUG
884-906
AD-53000.1
A-108401.1
UUUUGACUUGUAGUUUAUAUGUA
357-379
AD-53112.1
A-108591.1
AAAUCUUUGUUUUCCGGGAUUGC
1165-1187
AD-53107.1
A-108605.1
UUGCUUUGUGAUCCCAAGUAGAA
1194-1216
AD-53121.1
A-108641.1
AAAUUUGCCUCAGUUCAUUCAAA
1426-1448
AD-53046.1
A-108479.1
UUUCUUUUAUUUGACUAUGCUGU
624-646
AD-53038.1
A-108445.1
UAGUUAGUUGCUCUUCUAAAUAU
468-490
AD-53140.1
A-108663.1
UAUUGCCUUUUAAAUUUGCCUCA
1437-1459
AD-52987.1
A-108381.1
UAAAAAGACUGAUCAAAUAUGUU
277-299
AD-53130.1
A-108597.1
AAAACACCAAAUCUUUGUUUUCC
1173-1195
AD-53106.1
A-108589.1
AAUCUUUGUUUUCCGGGAUUGCA
1164-1186
AD-53081.1
A-108565.1
UAUUCAAUAUAAUGUUUGUUGUC
1069-1091
AD-53118.1
A-108593.1
AACACCAAAUCUUUGUUUUCCGG
1171-1193
AD-53136.1
A-108599.1
UAGAAAACACCAAAUCUUUGUUU
1176-1198
AD-53127.1
A-108643.1
UAAAUUUGCCUCAGUUCAUUCAA
1427-1449
AD-53066.1
A-108513.1
AUGUUGAAUUAAUGUCCAUGGAC
890-912
AD-53013.1
A-108421.1
UUUCAAGUUUUGAGUUGAGUUCA
405-427
AD-52991.1
A-108351.1
UUUAAGACCAUGUCCCAACUGAA
203-225
AD-53099.1
A-108571.1
AAGAAUAUUCAAUAUAAUGUUUG
1074-1096
AD-52958.1
A-108387.1
UUCUUCUUUGAUUUCACUGGUUU
314-336
AD-53097.1
A-108539.1
UAUAUCUUCUCUAGGCCCAACCA
991-1013
AD-52966.1
A-108327.1
AUCUUGAUUUUGGCUCUGGAGAU
138-160
AD-53145.1
A-108665.1
UUAUUGCCUUUUAAAUUUGCCUC
1438-1460
AD-53113.1
A-108607.1
UUUGCUUUGUGAUCCCAAGUAGA
1195-1217
AD-52993.1
A-108383.1
UAGAUCAUAAAAAGACUGAUCAA
284-306
AD-53031.1
A-108427.1
UUUUUCUUCUAGGAGGCUUUCAA
422-444
AD-53017.1
A-108485.1
UUCUAUUUCUUUUAUUUGACUAU
629-651
AD-53143.1
A-108633.1
UUGGUUGAUUUUAUAGAGUAUAA
1372-1394
AD-53149.1
A-108651.1
UUUUUAAAUUUGCCUCAGUUCAU
1431-
1453_G21A
AD-53059.1
A-108495.1
AACUUGAGAGUUGCUGGGUCUGA
836-858
AD-53006.1
A-108403.1
UUUUUGACUUGUAGUUUAUAUGU
358-380
AD-53025.1
A-108425.1
UUUUCUUCUAGGAGGCUUUCAAG
421-443
AD-53085.1
A-108535.1
AACCAUAUUUGUAGUUCUCCCAC
945-967
AD-52984.1
A-108333.1
AUCGUCUAACAUAGCAAAUCUUG
155-177
AD-53023.1
A-108487.1
UAGAGAAAUUUCUGUGGGUUCUU
677-699
AD-53014.1
A-108437.1
UAUUUCACUUUUUGUUGAAGUAG
448-470
AD-53060.1
A-108511.1
UUGAAUUAAUGUCCAUGGACUAC
887-909
AD-53110.1
A-108653.1
UCUUUUAAAUUUGCCUCAGUUCA
1432-
1454_G21A
AD-52980.1
A-108363.1
AAUAUGUCAUUAAUUUGGCCCUU
244-266
AD-53109.1
A-108637.1
UUCUGAAUCUGUUGGAUGGAUCA
1400-1422
AD-53141.1
A-108601.1
AAGUAGAAAACACCAAAUCUUUG
1179-1201
AD-53126.1
A-108627.1
UAUAACCUUCCAUUUUGAGACUU
1354-1376
AD-53116.1
A-108655.1
UCCUUUUAAAUUUGCCUCAGUUC
1433-
1455_C21A
AD-52997.1
A-108353.1
AAGUCUUUAAGACCAUGUCCCAA
208-230
AD-53120.1
A-108625.1
UUGGUUUGUUAUAUUUACCAUUU
1290-1312
AD-53070.1
A-108577.1
UAGUUGGUUUCGUGAUUUCCCAA
1102-1124
AD-53028.1
A-108473.1
UUUAUUUGACUAUGCUGUUGGUU
619-641
AD-53146.1
A-108603.1
UUUGUGAUCCCAAGUAGAAAACA
1190-1212
AD-52982.1
A-108395.1
UUGUAGUUUAUAUGUAGUUCUUC
350-372
AD-53111.1
A-108669.1
ACAGAUUUUUACACAUACUCUGU
1913-1935
AD-53045.1
A-108463.1
UUGAUGCUAUUAUCUUGUUUUUC
556-578
AD-53123.1
A-108673.1
UUACAGAUUUUUACACAUACUCU
1915-1937
AD-53018.1
A-108407.1
UUUACCUCUUCAUUUUUGACUUG
370-392
AD-52956.1
A-108355.1
AAAGUCUUUAAGACCAUGUCCCA
209-231
AD-53134.1
A-108661.1
AUUGCCUUUUAAAUUUGCCUCAG
1436-1458
AD-52968.1
A-108359.1
UAUGGACAAAGUCUUUAAGACCA
216-238
AD-53122.1
A-108657.1
UGCCUUUUAAAUUUGCCUCAGUU
1434-1456
AD-53100.1
A-108587.1
AUCUUUGUUUUCCGGGAUUGCAU
1163-1185
AD-53128.1
A-108659.1
UUGCCUUUUAAAUUUGCCUCAGU
1435-1457
AD-53043.1
A-108431.1
UUCACUUUUUGUUGAAGUAGAAU
445-467
AD-53135.1
A-108677.1
UAUUACAGAUUUUUACACAUACU
1917-1939
AD-53094.1
A-108585.1
UUUGUUUUCCGGGAUUGCAUUGG
1160-1182
AD-53019.1
A-108423.1
UUCUUCUAGGAGGCUUUCAAGUU
419-441
AD-53129.1
A-108675.1
AUUACAGAUUUUUACACAUACUC
1916-1938
AD-53150.1
A-108667.1
AAGAUUUUUACACAUACUCUGUG
1912-
1934_G21U
AD-53117.1
A-108671.1
UACAGAUUUUUACACAUACUCUG
1914-1936
AD-52985.1
A-108349.1
UUAAGACCAUGUCCCAACUGAAG
202-224
AD-52962.1
A-108357.1
AUGGACAAAGUCUUUAAGACCAU
215-237
AD-52974.1
A-108361.1
UUAUGGACAAAGUCUUUAAGACC
217-239
AD-52979.1
A-108347.1
UAAGACCAUGUCCCAACUGAAGG
201-223
The symbol “x” indicates that the sequence contains a GalNAc conjugate.
TABLE 8
Modified sense and antisense strand sequences of
ANGPTL3 GalNac-conjugated dsRNAs
Sense Sequence
Sense
(SEQ ID NOS 634-818,respectively, in
Duplex ID
OligoName
order of appearance)
AD-53063.1
A-108558.1
AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUfL96
AD-52965.1
A-108310.1
AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUfL96
AD-53030.1
A-108410.1
UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAfL96
AD-52953.1
A-108306.1
UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUfL96
AD-53001.1
A-108416.1
CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUfL96
AD-53080.1
A-108548.1
CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAfL96
AD-52971.1
A-108312.1
CfaAfuUfaAfgCfUfCfcUfuCfuUfuUfuAfL96
AD-53071.1
A-108498.1
AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUfL96
AD-53024.1
A-108408.1
GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAfL96
AD-52977.1
A-108314.1
AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUfL96
AD-53064.1
A-108574.1
CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUfL96
AD-53033.1
A-108458.1
AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAfL96
AD-52954.1
A-108322.1
UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUfL96
AD-53098.1
A-108554.1
CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAfL96
AD-53092.1
A-108552.1
CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUfL96
AD-53073.1
A-108530.1
GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAfL96
AD-53132.1
A-108628.1
AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAfL96
AD-53086.1
A-108550.1
UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUfL96
AD-52961.1
A-108340.1
CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAfL96
AD-52983.1
A-108316.1
AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUfL96
AD-53027.1
A-108456.1
AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAfL96
AD-52986.1
A-108364.1
GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUfL96
AD-52989.1
A-108318.1
UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUfL96
AD-52981.1
A-108378.1
AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96
AD-53077.1
A-108500.1
CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUfL96
AD-53095.1
A-108506.1
CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAfL96
AD-52970.1
A-108390.1
AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAfL96
AD-53015.1
A-108452.1
GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUfL96
AD-53147.1
A-108618.1
AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUfL96
AD-53103.1
A-108540.1
CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUfL96
AD-52969.1
A-108374.1
CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUfL96
AD-53075.1
A-108562.1
AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUfL96
AD-52994.1
A-108398.1
AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAfL96
AD-52960.1
A-108324.1
CfuAfgUfuAfuUfUfCfcUfcCfaGfaAfuUfL96
AD-53003.1
A-108448.1
AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAfL96
AD-52995.1
A-108320.1
UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUfL96
AD-53037.1
A-108428.1
CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAfL96
AD-53087.1
A-108566.1
AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUfL96
AD-53076.1
A-108578.1
GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUfL96
AD-52975.1
A-108376.1
AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUfL96
AD-53138.1
A-108630.1
UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAfL96
AD-53091.1
A-108536.1
GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUfL96
AD-53124.1
A-108594.1
GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUfL96
AD-53125.1
A-108610.1
AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAfL96
AD-53036.1
A-108412.1
GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAfL96
AD-53061.1
A-108526.1
GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUfL96
AD-53093.1
A-108568.1
AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUfL96
AD-53137.1
A-108614.1
UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUfL96
AD-52999.1
A-108384.1
AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUfL96
AD-53069.1
A-108560.1
GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAfL96
AD-53034.1
A-108474.1
CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAfL96
AD-52976.1
A-108392.1
CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAfL96
AD-52996.1
A-108336.1
UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAfL96
AD-53029.1
A-108488.1
AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUfL96
AD-53020.1
A-108438.1
CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUfL96
AD-53042.1
A-108414.1
UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAfL96
AD-53011.1
A-108482.1
CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAfL96
AD-52957.1
A-108370.1
CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUfL96
AD-53008.1
A-108434.1
UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUfL96
AD-53065.1
A-108496.1
GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUfL96
AD-53115.1
A-108638.1
UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUfL96
AD-53012.1
A-108404.1
UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUfL96
AD-53004.1
A-108464.1
AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAfL96
AD-53021.1
A-108454.1
CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAfL96
AD-52955.1
A-108338.1
GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAfL96
AD-53119.1
A-108608.1
AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAfL96
AD-52990.1
A-108334.1
UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAfL96
AD-52964.1
A-108388.1
AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAfL96
AD-52973.1
A-108344.1
GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUfL96
AD-53074.1
A-108546.1
AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAfL96
AD-53026.1
A-108440.1
UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUfL96
AD-53062.1
A-108542.1
CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAfL96
AD-53114.1
A-108622.1
CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAfL96
AD-53082.1
A-108580.1
GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAfL96
AD-53035.1
A-108490.1
CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUfL96
AD-52978.1
A-108330.1
AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAfL96
AD-53084.1
A-108518.1
AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAfL96
AD-52972.1
A-108328.1
CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUfL96
AD-53002.1
A-108432.1
CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAfL96
AD-53078.1
A-108516.1
GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUfL96
AD-53072.1
A-108514.1
GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAfL96
AD-53005.1
A-108480.1
GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUfL96
AD-53083.1
A-108502.1
CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAfL96
AD-53102.1
A-108524.1
AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAfL96
AD-53105.1
A-108572.1
AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUfL96
AD-53090.1
A-108520.1
UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUfL96
AD-53010.1
A-108466.1
GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUfL96
AD-52998.1
A-108368.1
UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAfL96
AD-52992.1
A-108366.1
AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAfL96
AD-53068.1
A-108544.1
GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAfL96
AD-53032.1
A-108442.1
AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAfL96
AD-52967.1
A-108342.1
CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAfL96
AD-53096.1
A-108522.1
UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUfL96
AD-53131.1
A-108612.1
GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAfL96
AD-52963.1
A-108372.1
UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUfL96
AD-53089.1
A-108504.1
UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAfL96
AD-53044.1
A-108446.1
UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAfL96
AD-52988.1
A-108396.1
UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAfL96
AD-53067.1
A-108528.1
GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAfL96
AD-53009.1
A-108450.1
AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUfL96
AD-53022.1
A-108470.1
AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAfL96
AD-53016.1
A-108468.1
AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAfL96
AD-53007.1
A-108418.1
GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAfL96
AD-53148.1
A-108634.1
UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAfL96
AD-53040.1
A-108476.1
CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAfL96
AD-53041.1
A-108492.1
GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUfL96
AD-53039.1
A-108460.1
CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUfL96
AD-53139.1
A-108646.1
AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAfL96
AD-53144.1
A-108648.1
UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAfL96
AD-53142.1
A-108616.1
AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAfL96
AD-53108.1
A-108620.1
AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAfL96
AD-53079.1
A-108532.1
AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAfL96
AD-53133.1
A-108644.1
AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAfL96
AD-53104.1
A-108556.1
GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAfL96
AD-53088.1
A-108582.1
UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUfL96
AD-53101.1
A-108508.1
GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUfL96
AD-53000.1
A-108400.1
CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAfL96
AD-53112.1
A-108590.1
AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUfL96
AD-53107.1
A-108604.1
CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAfL96
AD-53121.1
A-108640.1
UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUfL96
AD-53046.1
A-108478.1
AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAfL96
AD-53038.1
A-108444.1
AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAfL96
AD-53140.1
A-108662.1
AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAfL96
AD-52987.1
A-108380.1
CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAfL96
AD-53130.1
A-108596.1
AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUfL96
AD-53106.1
A-108588.1
CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUfL96
AD-53081.1
A-108564.1
CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAfL96
AD-53118.1
A-108592.1
GfgAfaAfaCfaAfAfGfaUfuUfgGfuGfuUfL96
AD-53136.1
A-108598.1
AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAfL96
AD-53127.1
A-108642.1
GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAfL96
AD-53066.1
A-108512.1
CfcAfuGfgAfcAfUfUfaAfuUfcAfaCfaUfL96
AD-53013.1
A-108420.1
AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAfL96
AD-52991.1
A-108350.1
CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAfL96
AD-53099.1
A-108570.1
AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUfL96
AD-52958.1
A-108386.1
AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAfL96
AD-53097.1
A-108538.1
GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAfL96
AD-52966.1
A-108326.1
CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUfL96
AD-53145.1
A-108664.1
GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAfL96
AD-53113.1
A-108606.1
UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAfL96
AD-52993.1
A-108382.1
GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAfL96
AD-53031.1
A-108426.1
GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAfL96
AD-53017.1
A-108484.1
AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAfL96
AD-53143.1
A-108632.1
AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAfL96
AD-53149.1
A-108650.1
GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAfL96
AD-53059.1
A-108494.1
AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUfL96
AD-53006.1
A-108402.1
AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAfL96
AD-53025.1
A-108424.1
UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAfL96
AD-53085.1
A-108534.1
GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUfL96
AD-52984.1
A-108332.1
AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUfL96
AD-53023.1
A-108486.1
GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAfL96
AD-53014.1
A-108436.1
AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAfL96
AD-53060.1
A-108510.1
AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAfL96
AD-53110.1
A-108652.1
AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAfL96
AD-52980.1
A-108362.1
GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUfL96
AD-53109.1
A-108636.1
AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAfL96
AD-53141.1
A-108600.1
AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUfL96
AD-53126.1
A-108626.1
GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAfL96
AD-53116.1
A-108654.1
AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAfL96
AD-52997.1
A-108352.1
GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUfL96
AD-53120.1
A-108624.1
AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAfL96
AD-53070.1
A-108576.1
GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAfL96
AD-53028.1
A-108472.1
CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAfL96
AD-53146.1
A-108602.1
UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAfL96
AD-52982.1
A-108394.1
AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAfL96
AD-53111.1
A-108668.1
AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUfL96
AD-53045.1
A-108462.1
AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAfL96
AD-53123.1
A-108672.1
AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAfL96
AD-53018.1
A-108406.1
AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAfL96
AD-52956.1
A-108354.1
GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUfL96
AD-53134.1
A-108660.1
GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUfL96
AD-52968.1
A-108358.1
GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAfL96
AD-53122.1
A-108656.1
CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAfL96
AD-53100.1
A-108586.1
GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUfL96
AD-53128.1
A-108658.1
UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAfL96
AD-53043.1
A-108430.1
UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAfL96
AD-53135.1
A-108676.1
UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAfL96
AD-53094.1
A-108584.1
AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAfL96
AD-53019.1
A-108422.1
CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAfL96
AD-53129.1
A-108674.1
GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUfL96
AD-53150.1
A-108666.1
CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUfL96
AD-53117.1
A-108670.1
GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAfL96
AD-52985.1
A-108348.1
UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAfL96
AD-52962.1
A-108356.1
GfgUfcUfuAfaAfGfAfcUfuUfgUfcCfaUfL96
AD-52974.1
A-108360.1
UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAfL96
AD-52979.1
A-108346.1
UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAfL96
Antisense Sequence
Antisense
(SEQ ID NOS 819-1003, respectively,
Duplex ID
OligoName
in order of appearance)
AD-53063.1
A-108559.1
aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc
AD-52965.1
A-108311.1
aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa
AD-53030.1
A-108411.1
uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa
AD-52953.1
A-108307.1
aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc
AD-53001.1
A-108417.1
aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg
AD-53080.1
A-108549.1
uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa
AD-52971.1
A-108313.1
uAfaAfaAfgAfaGfgagCfuUfaAfuUfgsUfsg
AD-53071.1
A-108499.1
aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu
AD-53024.1
A-108409.1
uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu
AD-52977.1
A-108315.1
aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu
AD-53064.1
A-108575.1
aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu
AD-53033.1
A-108459.1
uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg
AD-52954.1
A-108323.1
aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa
AD-53098.1
A-108555.1
uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa
AD-53092.1
A-108553.1
aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg
AD-53073.1
A-108531.1
uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa
AD-53132.1
A-108629.1
uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu
AD-53086.1
A-108551.1
aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu
AD-52961.1
A-108341.1
uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa
AD-52983.1
A-108317.1
aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg
AD-53027.1
A-108457.1
uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc
AD-52986.1
A-108365.1
aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu
AD-52989.1
A-108319.1
aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg
AD-52981.1
A-108379.1
aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg
AD-53077.1
A-108501.1
aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc
AD-53095.1
A-108507.1
uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu
AD-52970.1
A-108391.1
uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc
AD-53015.1
A-108453.1
aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu
AD-53147.1
A-108619.1
aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu
AD-53103.1
A-108541.1
aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc
AD-52969.1
A-108375.1
aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg
AD-53075.1
A-108563.1
aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu
AD-52994.1
A-108399.1
uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg
AD-52960.1
A-108325.1
aAfuUfcUfgGfaGfgaaAfuAfaCfuAfgsAfsg
AD-53003.1
A-108449.1
uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu
AD-52995.1
A-108321.1
aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa
AD-53037.1
A-108429.1
uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc
AD-53087.1
A-108567.1
aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu
AD-53076.1
A-108579.1
aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa
AD-52975.1
A-108377.1
aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa
AD-53138.1
A-108631.1
uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu
AD-53091.1
A-108537.1
aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa
AD-53124.1
A-108595.1
aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg
AD-53125.1
A-108611.1
uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa
AD-53036.1
A-108413.1
uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu
AD-53061.1
A-108527.1
aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa
AD-53093.1
A-108569.1
aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg
AD-53137.1
A-108615.1
aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu
AD-52999.1
A-108385.1
aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa
AD-53069.1
A-108561.1
uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu
AD-53034.1
A-108475.1
uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu
AD-52976.1
A-108393.1
uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu
AD-52996.1
A-108337.1
uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa
AD-53029.1
A-108489.1
aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu
AD-53020.1
A-108439.1
aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa
AD-53042.1
A-108415.1
uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa
AD-53011.1
A-108483.1
uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu
AD-52957.1
A-108371.1
aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa
AD-53008.1
A-108435.1
aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa
AD-53065.1
A-108497.1
aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg
AD-53115.1
A-108639.1
aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg
AD-53012.1
A-108405.1
aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg
AD-53004.1
A-108465.1
uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu
AD-53021.1
A-108455.1
uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu
AD-52955.1
A-108339.1
uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa
AD-53119.1
A-108609.1
uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg
AD-52990.1
A-108335.1
uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu
AD-52964.1
A-108389.1
uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc
AD-52973.1
A-108345.1
aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu
AD-53074.1
A-108547.1
uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu
AD-53026.1
A-108441.1
aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu
AD-53062.1
A-108543.1
uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc
AD-53114.1
A-108623.1
uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu
AD-53082.1
A-108581.1
uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc
AD-53035.1
A-108491.1
aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu
AD-52978.1
A-108331.1
uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg
AD-53084.1
A-108519.1
uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc
AD-52972.1
A-108329.1
aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg
AD-53002.1
A-108433.1
uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa
AD-53078.1
A-108517.1
aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa
AD-53072.1
A-108515.1
uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu
AD-53005.1
A-108481.1
aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg
AD-53083.1
A-108503.1
uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu
AD-53102.1
A-108525.1
uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu
AD-53105.1
A-108573.1
aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu
AD-53090.1
A-108521.1
aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg
AD-53010.1
A-108467.1
aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu
AD-52998.1
A-108369.1
uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu
AD-52992.1
A-108367.1
uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg
AD-53068.1
A-108545.1
uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc
AD-53032.1
A-108443.1
uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc
AD-52967.1
A-108343.1
uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc
AD-53096.1
A-108523.1
aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu
AD-53131.1
A-108613.1
uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc
AD-52963.1
A-108373.1
aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu
AD-53089.1
A-108505.1
uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa
AD-53044.1
A-108447.1
uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa
AD-52988.1
A-108397.1
uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu
AD-53067.1
A-108529.1
uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu
AD-53009.1
A-108451.1
aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc
AD-53022.1
A-108471.1
uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu
AD-53016.1
A-108469.1
uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa
AD-53007.1
A-108419.1
uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa
AD-53148.1
A-108635.1
uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa
AD-53040.1
A-108477.1
uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu
AD-53041.1
A-108493.1
aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu
AD-53039.1
A-108461.1
aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu
AD-53139.1
A-108647.1
uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc
AD-53144.1
A-108649.1
uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu
AD-53142.1
A-108617.1
uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc
AD-53108.1
A-108621.1
uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu
AD-53079.1
A-108533.1
uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc
AD-53133.1
A-108645.1
uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa
AD-53104.1
A-108557.1
uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc
AD-53088.1
A-108583.1
aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu
AD-53101.1
A-108509.1
aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg
AD-53000.1
A-108401.1
uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa
AD-53112.1
A-108591.1
aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc
AD-53107.1
A-108605.1
uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa
AD-53121.1
A-108641.1
aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa
AD-53046.1
A-108479.1
uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu
AD-53038.1
A-108445.1
uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu
AD-53140.1
A-108663.1
uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa
AD-52987.1
A-108381.1
uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu
AD-53130.1
A-108597.1
aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc
AD-53106.1
A-108589.1
aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa
AD-53081.1
A-108565.1
uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc
AD-53118.1
A-108593.1
aAfcAfcCfaAfaUfcuuUfgUfuUfuCfcsGfsg
AD-53136.1
A-108599.1
uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu
AD-53127.1
A-108643.1
uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa
AD-53066.1
A-108513.1
aUfgUfuGfaAfuUfaauGfuCfcAfuGfgsAfsc
AD-53013.1
A-108421.1
uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa
AD-52991.1
A-108351.1
uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa
AD-53099.1
A-108571.1
aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg
AD-52958.1
A-108387.1
uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu
AD-53097.1
A-108539.1
uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa
AD-52966.1
A-108327.1
aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu
AD-53145.1
A-108665.1
uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc
AD-53113.1
A-108607.1
uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa
AD-52993.1
A-108383.1
uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa
AD-53031.1
A-108427.1
uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa
AD-53017.1
A-108485.1
uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu
AD-53143.1
A-108633.1
uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa
AD-53149.1
A-108651.1
uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu
AD-53059.1
A-108495.1
aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa
AD-53006.1
A-108403.1
uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu
AD-53025.1
A-108425.1
uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg
AD-53085.1
A-108535.1
aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc
AD-52984.1
A-108333.1
aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg
AD-53023.1
A-108487.1
uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu
AD-53014.1
A-108437.1
uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg
AD-53060.1
A-108511.1
uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc
AD-53110.1
A-108653.1
uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa
AD-52980.1
A-108363.1
aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu
AD-53109.1
A-108637.1
uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa
AD-53141.1
A-108601.1
aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg
AD-53126.1
A-108627.1
uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu
AD-53116.1
A-108655.1
uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc
AD-52997.1
A-108353.1
aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa
AD-53120.1
A-108625.1
uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu
AD-53070.1
A-108577.1
uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa
AD-53028.1
A-108473.1
uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu
AD-53146.1
A-108603.1
uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa
AD-52982.1
A-108395.1
uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc
AD-53111.1
A-108669.1
aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu
AD-53045.1
A-108463.1
uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc
AD-53123.1
A-108673.1
uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu
AD-53018.1
A-108407.1
uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg
AD-52956.1
A-108355.1
aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa
AD-53134.1
A-108661.1
aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg
AD-52968.1
A-108359.1
uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa
AD-53122.1
A-108657.1
uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu
AD-53100.1
A-108587.1
aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu
AD-53128.1
A-108659.1
uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu
AD-53043.1
A-108431.1
uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu
AD-53135.1
A-108677.1
uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu
AD-53094.1
A-108585.1
uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg
AD-53019.1
A-108423.1
uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu
AD-53129.1
A-108675.1
aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc
AD-53150.1
A-108667.1
aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg
AD-53117.1
A-108671.1
uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg
AD-52985.1
A-108349.1
uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg
AD-52962.1
A-108357.1
aUfgGfaCfaAfaGfucuUfuAfaGfaCfcsAfsu
AD-52974.1
A-108361.1
uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc
AD-52979.1
A-108347.1
uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg
Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; Nf (e.g., Af) is a 2′-fluoro nucleotide; s is a phosphothiorate linkage; L96 indicates a GalNAc ligand.
TABLE 9
Unmodified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences are the same as the sequences
listed in Table 7 except that they do not contain
GalNal conjugation.
Sense Sequence
(SEQ ID NOS 1004-1184,
Sense
respectively, in order of
Antisense
Duplex Name
OligoName
appearance)
OligoName
AD-52637.1
A-108817.1
UCACAAUUAAGCUCCUUCUUU
A-108307.2
AD-52638.1
A-108825.1
UUAUUGUUCCUCUAGUUAUUU
A-108323.2
AD-52639.1
A-108833.1
GCUAUGUUAGACGAUGUAAAA
A-108339.2
AD-52640.1
A-108841.1
GGACAUGGUCUUAAAGACUUU
A-108355.2
AD-52641.1
A-108849.1
CAAAAACUCAACAUAUUUGAU
A-108371.2
AD-52642.1
A-108857.1
ACCAGUGAAAUCAAAGAAGAA
A-108387.2
AD-52643.1
A-108818.1
CACAAUUAAGCUCCUUCUUUU
A-108309.2
AD-52645.1
A-108834.1
CUAUGUUAGACGAUGUAAAAA
A-108341.2
AD-52647.1
A-108850.1
UCAACAUAUUUGAUCAGUCUU
A-108373.2
AD-52648.1
A-108858.1
AACUGAGAAGAACUACAUAUA
A-108389.2
AD-52649.1
A-108819.1
ACAAUUAAGCUCCUUCUUUUU
A-108311.2
AD-52650.1
A-108827.1
CUCCAGAGCCAAAAUCAAGAU
A-108327.2
AD-52651.1
A-108835.1
CGAUGUAAAAAUUUUAGCCAA
A-108343.2
AD-52652.1
A-108843.1
GUCUUAAAGACUUUGUCCAUA
A-108359.2
AD-52653.1
A-108851.1
CAACAUAUUUGAUCAGUCUUU
A-108375.2
AD-52654.1
A-108859.1
ACUGAGAAGAACUACAUAUAA
A-108391.2
AD-52656.1
A-108828.1
CCAGAGCCAAAAUCAAGAUUU
A-108329.2
AD-52657.1
A-108836.1
GAUGUAAAAAUUUUAGCCAAU
A-108345.2
AD-52658.1
A-108844.1
UCUUAAAGACUUUGUCCAUAA
A-108361.2
AD-52659.1
A-108852.1
AACAUAUUUGAUCAGUCUUUU
A-108377.2
AD-52660.1
A-108860.1
CUGAGAAGAACUACAUAUAAA
A-108393.2
AD-52661.1
A-108821.1
AAUUAAGCUCCUUCUUUUUAU
A-108315.2
AD-52662.1
A-108829.1
AAAUCAAGAUUUGCUAUGUUA
A-108331.2
AD-52663.1
A-108837.1
UUCAGUUGGGACAUGGUCUUA
A-108347.2
AD-52664.1
A-108845.1
GGGCCAAAUUAAUGACAUAUU
A-108363.2
AD-52665.1
A-108853.1
ACAUAUUUGAUCAGUCUUUUU
A-108379.2
AD-52666.1
A-108861.1
AGAACUACAUAUAAACUACAA
A-108395.2
AD-52667.1
A-108822.1
AUUAAGCUCCUUCUUUUUAUU
A-108317.2
AD-52668.1
A-108830.1
AGAUUUGCUAUGUUAGACGAU
A-108333.2
AD-52669.1
A-108838.1
UCAGUUGGGACAUGGUCUUAA
A-108349.2
AD-52670.1
A-108846.1
GGCCAAAUUAAUGACAUAUUU
A-108365.2
AD-52671.1
A-108854.1
CAUAUUUGAUCAGUCUUUUUA
A-108381.2
AD-52672.1
A-108862.1
UACAUAUAAACUACAAGUCAA
A-108397.2
AD-52673.1
A-108823.1
UUUUAUUGUUCCUCUAGUUAU
A-108319.2
AD-52674.1
A-108831.1
UUGCUAUGUUAGACGAUGUAA
A-108335.2
AD-52675.1
A-108839.1
CAGUUGGGACAUGGUCUUAAA
A-108351.2
AD-52676.1
A-108847.1
AAAUUAAUGACAUAUUUCAAA
A-108367.2
AD-52677.1
A-108855.1
GAUCAGUCUUUUUAUGAUCUA
A-108383.2
AD-52678.1
A-108863.1
ACAUAUAAACUACAAGUCAAA
A-108399.2
AD-52679.1
A-108824.1
UUUAUUGUUCCUCUAGUUAUU
A-108321.2
AD-52680.1
A-108832.1
UGCUAUGUUAGACGAUGUAAA
A-108337.2
AD-52681.1
A-108840.1
GGGACAUGGUCUUAAAGACUU
A-108353.2
AD-52682.1
A-108848.1
UGACAUAUUUCAAAAACUCAA
A-108369.2
AD-52683.1
A-108856.1
AUCAGUCUUUUUAUGAUCUAU
A-108385.2
AD-52684.1
A-108864.1
CAUAUAAACUACAAGUCAAAA
A-108401.2
AD-52685.1
A-108872.1
CUUGAACUCAACUCAAAACUU
A-108417.2
AD-52686.1
A-108880.1
CUACUUCAACAAAAAGUGAAA
A-108433.2
AD-52687.1
A-108888.1
AAGAGCAACUAACUAACUUAA
A-108449.2
AD-52688.1
A-108896.1
AAACAAGAUAAUAGCAUCAAA
A-108465.2
AD-52689.1
A-108904.1
GCAUAGUCAAAUAAAAGAAAU
A-108481.2
AD-52690.1
A-108865.1
AUAUAAACUACAAGUCAAAAA
A-108403.2
AD-52691.1
A-108873.1
GAACUCAACUCAAAACUUGAA
A-108419.2
AD-52692.1
A-108881.1
UACUUCAACAAAAAGUGAAAU
A-108435.2
AD-52693.1
A-108889.1
AGAGCAACUAACUAACUUAAU
A-108451.2
AD-52694.1
A-108897.1
GAUAAUAGCAUCAAAGACCUU
A-108467.2
AD-52695.1
A-108905.1
CAUAGUCAAAUAAAAGAAAUA
A-108483.2
AD-52696.1
A-108866.1
UAUAAACUACAAGUCAAAAAU
A-108405.2
AD-52697.1
A-108874.1
AACUCAACUCAAAACUUGAAA
A-108421.2
AD-52698.1
A-108882.1
ACUUCAACAAAAAGUGAAAUA
A-108437.2
AD-52699.1
A-108890.1
GAGCAACUAACUAACUUAAUU
A-108453.2
AD-52700.1
A-108898.1
AACCAACAGCAUAGUCAAAUA
A-108469.2
AD-52701.1
A-108906.1
AGUCAAAUAAAAGAAAUAGAA
A-108485.2
AD-52702.1
A-108867.1
AGUCAAAAAUGAAGAGGUAAA
A-108407.2
AD-52703.1
A-108875.1
CUUGAAAGCCUCCUAGAAGAA
A-108423.2
AD-52704.1
A-108883.1
CUUCAACAAAAAGUGAAAUAU
A-108439.2
AD-52705.1
A-108891.1
CAACUAACUAACUUAAUUCAA
A-108455.2
AD-52706.1
A-108899.1
ACCAACAGCAUAGUCAAAUAA
A-108471.2
AD-52707.1
A-108907.1
GAACCCACAGAAAUUUCUCUA
A-108487.2
AD-52708.1
A-108868.1
GAAUAUGUCACUUGAACUCAA
A-108409.2
AD-52709.1
A-108876.1
UGAAAGCCUCCUAGAAGAAAA
A-108425.2
AD-52710.1
A-108884.1
UUCAACAAAAAGUGAAAUAUU
A-108441.2
AD-52711.1
A-108892.1
AACUAACUAACUUAAUUCAAA
A-108457.2
AD-52712.1
A-108900.1
CCAACAGCAUAGUCAAAUAAA
A-108473.2
AD-52713.1
A-108908.1
AACCCACAGAAAUUUCUCUAU
A-108489.2
AD-52714.1
A-108869.1
UGUCACUUGAACUCAACUCAA
A-108411.2
AD-52715.1
A-108877.1
GAAAGCCUCCUAGAAGAAAAA
A-108427.2
AD-52716.1
A-108885.1
AAUAUUUAGAAGAGCAACUAA
A-108443.2
AD-52717.1
A-108893.1
ACUAACUAACUUAAUUCAAAA
A-108459.2
AD-52718.1
A-108901.1
CAACAGCAUAGUCAAAUAAAA
A-108475.2
AD-52719.1
A-108909.1
CCACAGAAAUUUCUCUAUCUU
A-108491.2
AD-52720.1
A-108870.1
GUCACUUGAACUCAACUCAAA
A-108413.2
AD-52721.1
A-108878.1
CUCCUAGAAGAAAAAAUUCUA
A-108429.2
AD-52722.1
A-108886.1
AUUUAGAAGAGCAACUAACUA
A-108445.2
AD-52723.1
A-108894.1
CUAACUAACUUAAUUCAAAAU
A-108461.2
AD-52724.1
A-108902.1
CAGCAUAGUCAAAUAAAAGAA
A-108477.2
AD-52725.1
A-108910.1
GAAAUAAGAAAUGUAAAACAU
A-108493.2
AD-52726.1
A-108871.1
UCACUUGAACUCAACUCAAAA
A-108415.2
AD-52727.1
A-108879.1
UCUACUUCAACAAAAAGUGAA
A-108431.2
AD-52728.1
A-108887.1
UUUAGAAGAGCAACUAACUAA
A-108447.2
AD-52729.1
A-108895.1
AAAACAAGAUAAUAGCAUCAA
A-108463.2
AD-52730.1
A-108903.1
AGCAUAGUCAAAUAAAAGAAA
A-108479.2
AD-52731.1
A-108958.1
AGACCCAGCAACUCUCAAGUU
A-108495.2
AD-52732.1
A-108966.1
AGUCCAUGGACAUUAAUUCAA
A-108511.2
AD-52733.1
A-108974.1
GAUGGAUCACAAAACUUCAAU
A-108527.2
AD-52734.1
A-108982.1
CUAGAGAAGAUAUACUCCAUA
A-108543.2
AD-52735.1
A-108990.1
AAAGACAACAAACAUUAUAUU
A-108559.2
AD-52736.1
A-108998.1
CAUUAUAUUGAAUAUUCUUUU
A-108575.2
AD-52737.1
A-108959.1
GACCCAGCAACUCUCAAGUUU
A-108497.2
AD-52739.1
A-108975.1
GGAUCACAAAACUUCAAUGAA
A-108529.2
AD-52740.1
A-108983.1
GAAGAUAUACUCCAUAGUGAA
A-108545.2
AD-52741.1
A-108991.1
GACAACAAACAUUAUAUUGAA
A-108561.2
AD-52742.1
A-108999.1
GGGAAAUCACGAAACCAACUA
A-108577.2
AD-52743.1
A-108960.1
ACCCAGCAACUCUCAAGUUUU
A-108499.2
AD-52744.1
A-108968.1
GGACAUUAAUUCAACAUCGAA
A-108515.2
AD-52745.1
A-108976.1
GAUCACAAAACUUCAAUGAAA
A-108531.2
AD-52746.1
A-108984.1
ACUCCAUAGUGAAGCAAUCUA
A-108547.2
AD-52747.1
A-108992.1
ACAACAAACAUUAUAUUGAAU
A-108563.2
AD-52748.1
A-109000.1
GGAAAUCACGAAACCAACUAU
A-108579.2
AD-52749.1
A-108961.1
CCCAGCAACUCUCAAGUUUUU
A-108501.2
AD-52750.1
A-108969.1
GACAUUAAUUCAACAUCGAAU
A-108517.2
AD-52751.1
A-108977.1
AACGUGGGAGAACUACAAAUA
A-108533.2
AD-52752.1
A-108985.1
CUCCAUAGUGAAGCAAUCUAA
A-108549.2
AD-52753.1
A-108993.1
CAACAAACAUUAUAUUGAAUA
A-108565.2
AD-52754.1
A-109001.1
GAAAUCACGAAACCAACUAUA
A-108581.2
AD-52755.1
A-108962.1
CUCUCAAGUUUUUCAUGUCUA
A-108503.2
AD-52756.1
A-108970.1
ACAUUAAUUCAACAUCGAAUA
A-108519.2
AD-52757.1
A-108978.1
GGGAGAACUACAAAUAUGGUU
A-108535.2
AD-52758.1
A-108986.1
UCCAUAGUGAAGCAAUCUAAU
A-108551.2
AD-52759.1
A-108994.1
AACAAACAUUAUAUUGAAUAU
A-108567.2
AD-52760.1
A-109002.1
UGGCAAUGUCCCCAAUGCAAU
A-108583.2
AD-52761.1
A-108963.1
UCAGGUAGUCCAUGGACAUUA
A-108505.2
AD-52762.1
A-108971.1
UUAAUUCAACAUCGAAUAGAU
A-108521.2
AD-52763.1
A-108979.1
GGAGAACUACAAAUAUGGUUU
A-108537.2
AD-52764.1
A-108987.1
CCAUAGUGAAGCAAUCUAAUU
A-108553.2
AD-52765.1
A-108995.1
ACAAACAUUAUAUUGAAUAUU
A-108569.2
AD-52766.1
A-109003.1
AAUGCAAUCCCGGAAAACAAA
A-108585.2
AD-52767.1
A-108964.1
CAGGUAGUCCAUGGACAUUAA
A-108507.2
AD-52768.1
A-108972.1
UUCAACAUCGAAUAGAUGGAU
A-108523.2
AD-52769.1
A-108980.1
GUUGGGCCUAGAGAAGAUAUA
A-108539.2
AD-52770.1
A-108988.1
CAUAGUGAAGCAAUCUAAUUA
A-108555.2
AD-52771.1
A-108996.1
AACAUUAUAUUGAAUAUUCUU
A-108571.2
AD-52772.1
A-109004.1
GCAAUCCCGGAAAACAAAGAU
A-108587.2
AD-52773.1
A-108965.1
GGUAGUCCAUGGACAUUAAUU
A-108509.2
AD-52774.1
A-108973.1
AUCGAAUAGAUGGAUCACAAA
A-108525.2
AD-52775.1
A-108981.1
CCUAGAGAAGAUAUACUCCAU
A-108541.2
AD-52776.1
A-108989.1
GUUGGAAGACUGGAAAGACAA
A-108557.2
AD-52777.1
A-108997.1
ACAUUAUAUUGAAUAUUCUUU
A-108573.2
AD-52778.1
A-109005.1
CAAUCCCGGAAAACAAAGAUU
A-108589.2
AD-52779.1
A-109013.1
CUACUUGGGAUCACAAAGCAA
A-108605.2
AD-52780.1
A-109021.1
ACAACCUAAAUGGUAAAUAUA
A-108621.2
AD-52781.1
A-109029.1
AUCCAUCCAACAGAUUCAGAA
A-108637.2
AD-52782.1
A-109037.1
AACUGAGGCAAAUUUAAAAGA
A-108653.2
AD-52783.1
A-109045.1
AGAGUAUGUGUAAAAAUCUGU
A-108669.2
AD-52784.1
A-109006.1
AAUCCCGGAAAACAAAGAUUU
A-108591.2
AD-52785.1
A-109014.1
UACUUGGGAUCACAAAGCAAA
A-108607.2
AD-52786.1
A-109022.1
CAACCUAAAUGGUAAAUAUAA
A-108623.2
AD-52787.1
A-109030.1
UUGAAUGAACUGAGGCAAAUU
A-108639.2
AD-52788.1
A-109038.1
ACUGAGGCAAAUUUAAAAGGA
A-108655.2
AD-52789.1
A-109046.1
GAGUAUGUGUAAAAAUCUGUA
A-108671.2
AD-52791.1
A-109015.1
ACUUGGGAUCACAAAGCAAAA
A-108609.2
AD-52792.1
A-109023.1
AUGGUAAAUAUAACAAACCAA
A-108625.2
AD-52793.1
A-109031.1
UGAAUGAACUGAGGCAAAUUU
A-108641.2
AD-52794.1
A-109039.1
CUGAGGCAAAUUUAAAAGGCA
A-108657.2
AD-52795.1
A-109047.1
AGUAUGUGUAAAAAUCUGUAA
A-108673.2
AD-52796.1
A-109008.1
GAAAACAAAGAUUUGGUGUUU
A-108595.2
AD-52797.1
A-109016.1
AGUGUGGAGAAAACAACCUAA
A-108611.2
AD-52798.1
A-109024.1
GUCUCAAAAUGGAAGGUUAUA
A-108627.2
AD-52799.1
A-109032.1
GAAUGAACUGAGGCAAAUUUA
A-108643.2
AD-52800.1
A-109040.1
UGAGGCAAAUUUAAAAGGCAA
A-108659.2
AD-52801.1
A-109048.1
GUAUGUGUAAAAAUCUGUAAU
A-108675.2
AD-52802.1
A-109009.1
AAAACAAAGAUUUGGUGUUUU
A-108597.2
AD-52803.1
A-109017.1
GUGUGGAGAAAACAACCUAAA
A-108613.2
AD-52804.1
A-109025.1
AUGGAAGGUUAUACUCUAUAA
A-108629.2
AD-52805.1
A-109033.1
AAUGAACUGAGGCAAAUUUAA
A-108645.2
AD-52806.1
A-109041.1
GAGGCAAAUUUAAAAGGCAAU
A-108661.2
AD-52807.1
A-109049.1
UAUGUGUAAAAAUCUGUAAUA
A-108677.2
AD-52808.1
A-109010.1
ACAAAGAUUUGGUGUUUUCUA
A-108599.2
AD-52809.1
A-109018.1
UGUGGAGAAAACAACCUAAAU
A-108615.2
AD-52810.1
A-109026.1
UGGAAGGUUAUACUCUAUAAA
A-108631.2
AD-52811.1
A-109034.1
AUGAACUGAGGCAAAUUUAAA
A-108647.2
AD-52812.1
A-109042.1
AGGCAAAUUUAAAAGGCAAUA
A-108663.2
AD-52813.1
A-109011.1
AAGAUUUGGUGUUUUCUACUU
A-108601.2
AD-52814.1
A-109019.1
AAACAACCUAAAUGGUAAAUA
A-108617.2
AD-52815.1
A-109027.1
AUACUCUAUAAAAUCAACCAA
A-108633.2
AD-52816.1
A-109035.1
UGAACUGAGGCAAAUUUAAAA
A-108649.2
AD-52817.1
A-109043.1
GGCAAAUUUAAAAGGCAAUAA
A-108665.2
AD-52818.1
A-109012.1
UUUUCUACUUGGGAUCACAAA
A-108603.2
AD-52819.1
A-109020.1
AACAACCUAAAUGGUAAAUAU
A-108619.2
AD-52820.1
A-109028.1
UACUCUAUAAAAUCAACCAAA
A-108635.2
AD-52821.1
A-109036.1
GAACUGAGGCAAAUUUAAAAA
A-108651.2
AD-52822.1
A-109044.1
CAGAGUAUGUGUAAAAAUCUU
A-108667.2
Antisense Sequence
(SEQ ID NOS 1185-1365,
respectively, in order of
Position in
Duplex Name
appearance)
NM_014495.2
AD-52637.1
AAAGAAGGAGCUUAAUUGUGAAC
54-76
AD-52638.1
AAAUAACUAGAGGAACAAUAAAA
75-97
AD-52639.1
UUUUACAUCGUCUAACAUAGCAA
161-183
AD-52640.1
AAAGUCUUUAAGACCAUGUCCCA
209-231
AD-52641.1
AUCAAAUAUGUUGAGUUUUUGAA
266-288
AD-52642.1
UUCUUCUUUGAUUUCACUGGUUU
314-336
AD-52643.1
AAAAGAAGGAGCUUAAUUGUGAA
55-77
AD-52645.1
UUUUUACAUCGUCUAACAUAGCA
162-184
AD-52647.1
AAGACUGAUCAAAUAUGUUGAGU
273-295
AD-52648.1
UAUAUGUAGUUCUUCUCAGUUCC
342-364
AD-52649.1
AAAAAGAAGGAGCUUAAUUGUGA
56-78
AD-52650.1
AUCUUGAUUUUGGCUCUGGAGAU
138-160
AD-52651.1
UUGGCUAAAAUUUUUACAUCGUC
172-194
AD-52652.1
UAUGGACAAAGUCUUUAAGACCA
216-238
AD-52653.1
AAAGACUGAUCAAAUAUGUUGAG
274-296
AD-52654.1
UUAUAUGUAGUUCUUCUCAGUUC
343-365
AD-52656.1
AAAUCUUGAUUUUGGCUCUGGAG
140-162
AD-52657.1
AUUGGCUAAAAUUUUUACAUCGU
173-195
AD-52658.1
UUAUGGACAAAGUCUUUAAGACC
217-239
AD-52659.1
AAAAGACUGAUCAAAUAUGUUGA
275-297
AD-52660.1
UUUAUAUGUAGUUCUUCUCAGUU
344-366
AD-52661.1
AUAAAAAGAAGGAGCUUAAUUGU
58-80
AD-52662.1
UAACAUAGCAAAUCUUGAUUUUG
149-171
AD-52663.1
UAAGACCAUGUCCCAACUGAAGG
201-223
AD-52664.1
AAUAUGUCAUUAAUUUGGCCCUU
244-266
AD-52665.1
AAAAAGACUGAUCAAAUAUGUUG
276-298
AD-52666.1
UUGUAGUUUAUAUGUAGUUCUUC
350-372
AD-52667.1
AAUAAAAAGAAGGAGCUUAAUUG
59-81
AD-52668.1
AUCGUCUAACAUAGCAAAUCUUG
155-177
AD-52669.1
UUAAGACCAUGUCCCAACUGAAG
202-224
AD-52670.1
AAAUAUGUCAUUAAUUUGGCCCU
245-267
AD-52671.1
UAAAAAGACUGAUCAAAUAUGUU
277-299
AD-52672.1
UUGACUUGUAGUUUAUAUGUAGU
355-377
AD-52673.1
AUAACUAGAGGAACAAUAAAAAG
73-95
AD-52674.1
UUACAUCGUCUAACAUAGCAAAU
159-181
AD-52675.1
UUUAAGACCAUGUCCCAACUGAA
203-225
AD-52676.1
UUUGAAAUAUGUCAUUAAUUUGG
249-271
AD-52677.1
UAGAUCAUAAAAAGACUGAUCAA
284-306
AD-52678.1
UUUGACUUGUAGUUUAUAUGUAG
356-378
AD-52679.1
AAUAACUAGAGGAACAAUAAAAA
74-96
AD-52680.1
UUUACAUCGUCUAACAUAGCAAA
160-182
AD-52681.1
AAGUCUUUAAGACCAUGUCCCAA
208-230
AD-52682.1
UUGAGUUUUUGAAAUAUGUCAUU
256-278
AD-52683.1
AUAGAUCAUAAAAAGACUGAUCA
285-307
AD-52684.1
UUUUGACUUGUAGUUUAUAUGUA
357-379
AD-52685.1
AAGUUUUGAGUUGAGUUCAAGUG
401-423
AD-52686.1
UUUCACUUUUUGUUGAAGUAGAA
446-468
AD-52687.1
UUAAGUUAGUUAGUUGCUCUUCU
474-496
AD-52688.1
UUUGAUGCUAUUAUCUUGUUUUU
557-579
AD-52689.1
AUUUCUUUUAUUUGACUAUGCUG
625-647
AD-52690.1
UUUUUGACUUGUAGUUUAUAUGU
358-380
AD-52691.1
UUCAAGUUUUGAGUUGAGUUCAA
404-426
AD-52692.1
AUUUCACUUUUUGUUGAAGUAGA
447-469
AD-52693.1
AUUAAGUUAGUUAGUUGCUCUUC
475-497
AD-52694.1
AAGGUCUUUGAUGCUAUUAUCUU
563-585
AD-52695.1
UAUUUCUUUUAUUUGACUAUGCU
626-648
AD-52696.1
AUUUUUGACUUGUAGUUUAUAUG
359-381
AD-52697.1
UUUCAAGUUUUGAGUUGAGUUCA
405-427
AD-52698.1
UAUUUCACUUUUUGUUGAAGUAG
448-470
AD-52699.1
AAUUAAGUUAGUUAGUUGCUCUU
476-498
AD-52700.1
UAUUUGACUAUGCUGUUGGUUUA
617-639
AD-52701.1
UUCUAUUUCUUUUAUUUGACUAU
629-651
AD-52702.1
UUUACCUCUUCAUUUUUGACUUG
370-392
AD-52703.1
UUCUUCUAGGAGGCUUUCAAGUU
419-441
AD-52704.1
AUAUUUCACUUUUUGUUGAAGUA
449-471
AD-52705.1
UUGAAUUAAGUUAGUUAGUUGCU
479-501
AD-52706.1
UUAUUUGACUAUGCUGUUGGUUU
618-640
AD-52707.1
UAGAGAAAUUUCUGUGGGUUCUU
677-699
AD-52708.1
UUGAGUUCAAGUGACAUAUUCUU
391-413
AD-52709.1
UUUUCUUCUAGGAGGCUUUCAAG
421-443
AD-52710.1
AAUAUUUCACUUUUUGUUGAAGU
450-472
AD-52711.1
UUUGAAUUAAGUUAGUUAGUUGC
480-502
AD-52712.1
UUUAUUUGACUAUGCUGUUGGUU
619-641
AD-52713.1
AUAGAGAAAUUUCUGUGGGUUCU
678-700
AD-52714.1
UUGAGUUGAGUUCAAGUGACAUA
396-418
AD-52715.1
UUUUUCUUCUAGGAGGCUUUCAA
422-444
AD-52716.1
UUAGUUGCUCUUCUAAAUAUUUC
465-487
AD-52717.1
UUUUGAAUUAAGUUAGUUAGUUG
481-503
AD-52718.1
UUUUAUUUGACUAUGCUGUUGGU
620-642
AD-52719.1
AAGAUAGAGAAAUUUCUGUGGGU
681-703
AD-52720.1
UUUGAGUUGAGUUCAAGUGACAU
397-419
AD-52721.1
UAGAAUUUUUUCUUCUAGGAGGC
428-450
AD-52722.1
UAGUUAGUUGCUCUUCUAAAUAU
468-490
AD-52723.1
AUUUUGAAUUAAGUUAGUUAGUU
482-504
AD-52724.1
UUCUUUUAUUUGACUAUGCUGUU
623-645
AD-52725.1
AUGUUUUACAUUUCUUAUUUCAU
746-768
AD-52726.1
UUUUGAGUUGAGUUCAAGUGACA
398-420
AD-52727.1
UUCACUUUUUGUUGAAGUAGAAU
445-467
AD-52728.1
UUAGUUAGUUGCUCUUCUAAAUA
469-491
AD-52729.1
UUGAUGCUAUUAUCUUGUUUUUC
556-578
AD-52730.1
UUUCUUUUAUUUGACUAUGCUGU
624-646
AD-52731.1
AACUUGAGAGUUGCUGGGUCUGA
836-858
AD-52732.1
UUGAAUUAAUGUCCAUGGACUAC
887-909
AD-52733.1
AUUGAAGUUUUGUGAUCCAUCUA
917-939
AD-52734.1
UAUGGAGUAUAUCUUCUCUAGGC
998-1020
AD-52735.1
AAUAUAAUGUUUGUUGUCUUUCC
1064-1086
AD-52736.1
AAAAGAAUAUUCAAUAUAAUGUU
1076-1098
AD-52737.1
AAACUUGAGAGUUGCUGGGUCUG
837-859
AD-52739.1
UUCAUUGAAGUUUUGUGAUCCAU
920-942
AD-52740.1
UUCACUAUGGAGUAUAUCUUCUC
1003-1025
AD-52741.1
UUCAAUAUAAUGUUUGUUGUCUU
1067-1089
AD-52742.1
UAGUUGGUUUCGUGAUUUCCCAA
1102-1124
AD-52743.1
AAAACUUGAGAGUUGCUGGGUCU
838-860
AD-52744.1
UUCGAUGUUGAAUUAAUGUCCAU
894-916
AD-52745.1
UUUCAUUGAAGUUUUGUGAUCCA
921-943
AD-52746.1
UAGAUUGCUUCACUAUGGAGUAU
1011-1033
AD-52747.1
AUUCAAUAUAAUGUUUGUUGUCU
1068-1090
AD-52748.1
AUAGUUGGUUUCGUGAUUUCCCA
1103-1125
AD-52749.1
AAAAACUUGAGAGUUGCUGGGUC
839-861
AD-52750.1
AUUCGAUGUUGAAUUAAUGUCCA
895-917
AD-52751.1
UAUUUGUAGUUCUCCCACGUUUC
940-962
AD-52752.1
UUAGAUUGCUUCACUAUGGAGUA
1012-1034
AD-52753.1
UAUUCAAUAUAAUGUUUGUUGUC
1069-1091
AD-52754.1
UAUAGUUGGUUUCGUGAUUUCCC
1104-1126
AD-52755.1
UAGACAUGAAAAACUUGAGAGUU
847-869
AD-52756.1
UAUUCGAUGUUGAAUUAAUGUCC
896-918
AD-52757.1
AACCAUAUUUGUAGUUCUCCCAC
945-967
AD-52758.1
AUUAGAUUGCUUCACUAUGGAGU
1013-1035
AD-52759.1
AUAUUCAAUAUAAUGUUUGUUGU
1070-1092
AD-52760.1
AUUGCAUUGGGGACAUUGCCAGU
1147-1169
AD-52761.1
UAAUGUCCAUGGACUACCUGAUA
881-903
AD-52762.1
AUCUAUUCGAUGUUGAAUUAAUG
899-921
AD-52763.1
AAACCAUAUUUGUAGUUCUCCCA
946-968
AD-52764.1
AAUUAGAUUGCUUCACUAUGGAG
1014-1036
AD-52765.1
AAUAUUCAAUAUAAUGUUUGUUG
1071-1093
AD-52766.1
UUUGUUUUCCGGGAUUGCAUUGG
1160-1182
AD-52767.1
UUAAUGUCCAUGGACUACCUGAU
882-904
AD-52768.1
AUCCAUCUAUUCGAUGUUGAAUU
903-925
AD-52769.1
UAUAUCUUCUCUAGGCCCAACCA
991-1013
AD-52770.1
UAAUUAGAUUGCUUCACUAUGGA
1015-1037
AD-52771.1
AAGAAUAUUCAAUAUAAUGUUUG
1074-1096
AD-52772.1
AUCUUUGUUUUCCGGGAUUGCAU
1163-1185
AD-52773.1
AAUUAAUGUCCAUGGACUACCUG
884-906
AD-52774.1
UUUGUGAUCCAUCUAUUCGAUGU
909-931
AD-52775.1
AUGGAGUAUAUCUUCUCUAGGCC
997-1019
AD-52776.1
UUGUCUUUCCAGUCUUCCAACUC
1051-1073
AD-52777.1
AAAGAAUAUUCAAUAUAAUGUUU
1075-1097
AD-52778.1
AAUCUUUGUUUUCCGGGAUUGCA
1164-1186
AD-52779.1
UUGCUUUGUGAUCCCAAGUAGAA
1194-1216
AD-52780.1
UAUAUUUACCAUUUAGGUUGUUU
1281-1303
AD-52781.1
UUCUGAAUCUGUUGGAUGGAUCA
1400-1422
AD-52782.1
UCUUUUAAAUUUGCCUCAGUUCA
1432-
1454_G21A
AD-52783.1
ACAGAUUUUUACACAUACUCUGU
1913-1935
AD-52784.1
AAAUCUUUGUUUUCCGGGAUUGC
1165-1187
AD-52785.1
UUUGCUUUGUGAUCCCAAGUAGA
1195-1217
AD-52786.1
UUAUAUUUACCAUUUAGGUUGUU
1282-1304
AD-52787.1
AAUUUGCCUCAGUUCAUUCAAAG
1425-1447
AD-52788.1
UCCUUUUAAAUUUGCCUCAGUUC
1433-
1455_C21A
AD-52789.1
UACAGAUUUUUACACAUACUCUG
1914-1936
AD-52791.1
UUUUGCUUUGUGAUCCCAAGUAG
1196-1218
AD-52792.1
UUGGUUUGUUAUAUUUACCAUUU
1290-1312
AD-52793.1
AAAUUUGCCUCAGUUCAUUCAAA
1426-1448
AD-52794.1
UGCCUUUUAAAUUUGCCUCAGUU
1434-1456
AD-52795.1
UUACAGAUUUUUACACAUACUCU
1915-1937
AD-52796.1
AAACACCAAAUCUUUGUUUUCCG
1172-1194
AD-52797.1
UUAGGUUGUUUUCUCCACACUCA
1269-1291
AD-52798.1
UAUAACCUUCCAUUUUGAGACUU
1354-1376
AD-52799.1
UAAAUUUGCCUCAGUUCAUUCAA
1427-1449
AD-52800.1
UUGCCUUUUAAAUUUGCCUCAGU
1435-1457
AD-52801.1
AUUACAGAUUUUUACACAUACUC
1916-1938
AD-52802.1
AAAACACCAAAUCUUUGUUUUCC
1173-1195
AD-52803.1
UUUAGGUUGUUUUCUCCACACUC
1270-1292
AD-52804.1
UUAUAGAGUAUAACCUUCCAUUU
1362-1384
AD-52805.1
UUAAAUUUGCCUCAGUUCAUUCA
1428-1450
AD-52806.1
AUUGCCUUUUAAAUUUGCCUCAG
1436-1458
AD-52807.1
UAUUACAGAUUUUUACACAUACU
1917-1939
AD-52808.1
UAGAAAACACCAAAUCUUUGUUU
1176-1198
AD-52809.1
AUUUAGGUUGUUUUCUCCACACU
1271-1293
AD-52810.1
UUUAUAGAGUAUAACCUUCCAUU
1363-1385
AD-52811.1
UUUAAAUUUGCCUCAGUUCAUUC
1429-1451
AD-52812.1
UAUUGCCUUUUAAAUUUGCCUCA
1437-1459
AD-52813.1
AAGUAGAAAACACCAAAUCUUUG
1179-1201
AD-52814.1
UAUUUACCAUUUAGGUUGUUUUC
1279-1301
AD-52815.1
UUGGUUGAUUUUAUAGAGUAUAA
1372-1394
AD-52816.1
UUUUAAAUUUGCCUCAGUUCAUU
1430-1452
AD-52817.1
UUAUUGCCUUUUAAAUUUGCCUC
1438-1460
AD-52818.1
UUUGUGAUCCCAAGUAGAAAACA
1190-1212
AD-52819.1
AUAUUUACCAUUUAGGUUGUUUU
1280-1302
AD-52820.1
UUUGGUUGAUUUUAUAGAGUAUA
1373-1395
AD-52821.1
UUUUUAAAUUUGCCUCAGUUCAU
1431-
1453_G21A
AD-52822.1
AAGAUUUUUACACAUACUCUGUG
1912-
1934_G21U
TABLE 10
Modified Sense and antisense strand sequences
of ANGPTL3 dsRNAs without GalNal conjugation
These sequences are the same as the sequences listed
in Table 8 except that they do not contain GalNal
conjugation.
Sense Sequence
Duplex
Sense Oligo
(SEQ ID NOS 1366-1546, respectively,
Name
Name
in order of appearance)
AD-52637.1
A-108817.1
UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUf
AD-52638.1
A-108825.1
UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf
AD-52639.1
A-108833.1
GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf
AD-52640.1
A-108841.1
GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf
AD-52641.1
A-108849.1
CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf
AD-52642.1
A-108857.1
AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf
AD-52643.1
A-108818.1
CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf
AD-52645.1
A-108834.1
CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf
AD-52647.1
A-108850.1
UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf
AD-52648.1
A-108858.1
AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf
AD-52649.1
A-108819.1
AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUf
AD-52650.1
A-108827.1
CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf
AD-52651.1
A-108835.1
CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf
AD-52652.1
A-108843.1
GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf
AD-52653.1
A-108851.1
CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf
AD-52654.1
A-108859.1
AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAf
AD-52656.1
A-108828.1
CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUf
AD-52657.1
A-108836.1
GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf
AD-52658.1
A-108844.1
UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf
AD-52659.1
A-108852.1
AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf
AD-52660.1
A-108860.1
CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf
AD-52661.1
A-108821.1
AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUf
AD-52662.1
A-108829.1
AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf
AD-52663.1
A-108837.1
UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf
AD-52664.1
A-108845.1
GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUf
AD-52665.1
A-108853.1
AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf
AD-52666.1
A-108861.1
AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAf
AD-52667.1
A-108822.1
AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUf
AD-52668.1
A-108830.1
AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUf
AD-52669.1
A-108838.1
UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAf
AD-52670.1
A-108846.1
GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUf
AD-52671.1
A-108854.1
CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAf
AD-52672.1
A-108862.1
UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAf
AD-52673.1
A-108823.1
UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUf
AD-52674.1
A-108831.1
UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAf
AD-52675.1
A-108839.1
CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAf
AD-52676.1
A-108847.1
AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAf
AD-52677.1
A-108855.1
GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAf
AD-52678.1
A-108863.1
AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAf
AD-52679.1
A-108824.1
UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUf
AD-52680.1
A-108832.1
UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAf
AD-52681.1
A-108840.1
GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUf
AD-52682.1
A-108848.1
UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAf
AD-52683.1
A-108856.1
AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUf
AD-52684.1
A-108864.1
CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAf
AD-52685.1
A-108872.1
CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUf
AD-52686.1
A-108880.1
CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAf
AD-52687.1
A-108888.1
AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAf
AD-52688.1
A-108896.1
AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAf
AD-52689.1
A-108904.1
GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUf
AD-52690.1
A-108865.1
AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAf
AD-52691.1
A-108873.1
GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAf
AD-52692.1
A-108881.1
UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUf
AD-52693.1
A-108889.1
AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUf
AD-52694.1
A-108897.1
GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUf
AD-52695.1
A-108905.1
CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAf
AD-52696.1
A-108866.1
UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUf
AD-52697.1
A-108874.1
AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAf
AD-52698.1
A-108882.1
AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAf
AD-52699.1
A-108890.1
GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf
AD-52700.1
A-108898.1
AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAf
AD-52701.1
A-108906.1
AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf
AD-52702.1
A-108867.1
AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf
AD-52703.1
A-108875.1
CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAf
AD-52704.1
A-108883.1
CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUf
AD-52705.1
A-108891.1
CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAf
AD-52706.1
A-108899.1
AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAf
AD-52707.1
A-108907.1
GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAf
AD-52708.1
A-108868.1
GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAf
AD-52709.1
A-108876.1
UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAf
AD-52710.1
A-108884.1
UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUf
AD-52711.1
A-108892.1
AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAf
AD-52712.1
A-108900.1
CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAf
AD-52713.1
A-108908.1
AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUf
AD-52714.1
A-108869.1
UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf
AD-52715.1
A-108877.1
GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAf
AD-52716.1
A-108885.1
AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAf
AD-52717.1
A-108893.1
AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAf
AD-52718.1
A-108901.1
CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf
AD-52719.1
A-108909.1
CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUf
AD-52720.1
A-108870.1
GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf
AD-52721.1
A-108878.1
CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAf
AD-52722.1
A-108886.1
AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAf
AD-52723.1
A-108894.1
CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUf
AD-52724.1
A-108902.1
CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAf
AD-52725.1
A-108910.1
GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUf
AD-52726.1
A-108871.1
UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAf
AD-52727.1
A-108879.1
UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAf
AD-52728.1
A-108887.1
UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAf
AD-52729.1
A-108895.1
AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAf
AD-52730.1
A-108903.1
AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAf
AD-52731.1
A-108958.1
AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUf
AD-52732.1
A-108966.1
AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAf
AD-52733.1
A-108974.1
GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUf
AD-52734.1
A-108982.1
CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAf
AD-52735.1
A-108990.1
AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf
AD-52736.1
A-108998.1
CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUf
AD-52737.1
A-108959.1
GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUf
AD-52739.1
A-108975.1
GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAf
AD-52740.1
A-108983.1
GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAf
AD-52741.1
A-108991.1
GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf
AD-52742.1
A-108999.1
GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAf
AD-52743.1
A-108960.1
AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUf
AD-52744.1
A-108968.1
GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAf
AD-52745.1
A-108976.1
GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAf
AD-52746.1
A-108984.1
AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAf
AD-52747.1
A-108992.1
AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf
AD-52748.1
A-109000.1
GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUf
AD-52749.1
A-108961.1
CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUf
AD-52750.1
A-108969.1
GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUf
AD-52751.1
A-108977.1
AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAf
AD-52752.1
A-108985.1
CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAf
AD-52753.1
A-108993.1
CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAf
AD-52754.1
A-109001.1
GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAf
AD-52755.1
A-108962.1
CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAf
AD-52756.1
A-108970.1
AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAf
AD-52757.1
A-108978.1
GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUf
AD-52758.1
A-108986.1
UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUf
AD-52759.1
A-108994.1
AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUf
AD-52760.1
A-109002.1
UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUf
AD-52761.1
A-108963.1
UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAf
AD-52762.1
A-108971.1
UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUf
AD-52763.1
A-108979.1
GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUf
AD-52764.1
A-108987.1
CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUf
AD-52765.1
A-108995.1
AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUf
AD-52766.1
A-109003.1
AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAf
AD-52767.1
A-108964.1
CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAf
AD-52768.1
A-108972.1
UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUf
AD-52769.1
A-108980.1
GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAf
AD-52770.1
A-108988.1
CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAf
AD-52771.1
A-108996.1
AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf
AD-52772.1
A-109004.1
GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUf
AD-52773.1
A-108965.1
GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUf
AD-52774.1
A-108973.1
AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAf
AD-52775.1
A-108981.1
CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUf
AD-52776.1
A-108989.1
GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAf
AD-52777.1
A-108997.1
AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUf
AD-52778.1
A-109005.1
CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUf
AD-52779.1
A-109013.1
CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAf
AD-52780.1
A-109021.1
AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAf
AD-52781.1
A-109029.1
AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAf
AD-52782.1
A-109037.1
AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAf
AD-52783.1
A-109045.1
AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUf
AD-52784.1
A-109006.1
AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUf
AD-52785.1
A-109014.1
UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAf
AD-52786.1
A-109022.1
CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAf
AD-52787.1
A-109030.1
UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUf
AD-52788.1
A-109038.1
AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAf
AD-52789.1
A-109046.1
GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAf
AD-52791.1
A-109015.1
AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAf
AD-52792.1
A-109023.1
AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAf
AD-52793.1
A-109031.1
UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUf
AD-52794.1
A-109039.1
CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAf
AD-52795.1
A-109047.1
AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAf
AD-52796.1
A-109008.1
GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUf
AD-52797.1
A-109016.1
AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAf
AD-52798.1
A-109024.1
GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAf
AD-52799.1
A-109032.1
GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAf
AD-52800.1
A-109040.1
UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAf
AD-52801.1
A-109048.1
GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUf
AD-52802.1
A-109009.1
AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUf
AD-52803.1
A-109017.1
GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAf
AD-52804.1
A-109025.1
AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAf
AD-52805.1
A-109033.1
AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAf
AD-52806.1
A-109041.1
GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUf
AD-52807.1
A-109049.1
UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAf
AD-52808.1
A-109010.1
AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAf
AD-52809.1
A-109018.1
UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUf
AD-52810.1
A-109026.1
UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAf
AD-52811.1
A-109034.1
AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAf
AD-52812.1
A-109042.1
AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAf
AD-52813.1
A-109011.1
AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUf
AD-52814.1
A-109019.1
AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAf
AD-52815.1
A-109027.1
AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAf
AD-52816.1
A-109035.1
UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAf
AD-52817.1
A-109043.1
GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAf
AD-52818.1
A-109012.1
UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAf
AD-52819.1
A-109020.1
AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUf
AD-52820.1
A-109028.1
UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAf
AD-52821.1
A-109036.1
GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAf
AD-52822.1
A-109044.1
CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUf
Antisense Oligo Sequence
Duplex
Antisense
(SEQ ID NOS 1547-1727, respectively,
Name
OligoName
in order of appearance)
AD-52637.1
A-108307.2
aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc
AD-52638.1
A-108323.2
aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa
AD-52639.1
A-108339.2
uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa
AD-52640.1
A-108355.2
aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa
AD-52641.1
A-108371.2
aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa
AD-52642.1
A-108387.2
uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu
AD-52643.1
A-108309.2
aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa
AD-52645.1
A-108341.2
uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa
AD-52647.1
A-108373.2
aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu
AD-52648.1
A-108389.2
uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc
AD-52649.1
A-108311.2
aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa
AD-52650.1
A-108327.2
aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu
AD-52651.1
A-108343.2
uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc
AD-52652.1
A-108359.2
uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa
AD-52653.1
A-108375.2
aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg
AD-52654.1
A-108391.2
uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc
AD-52656.1
A-108329.2
aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg
AD-52657.1
A-108345.2
aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu
AD-52658.1
A-108361.2
uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc
AD-52659.1
A-108377.2
aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa
AD-52660.1
A-108393.2
uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu
AD-52661.1
A-108315.2
aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu
AD-52662.1
A-108331.2
uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg
AD-52663.1
A-108347.2
uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg
AD-52664.1
A-108363.2
aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu
AD-52665.1
A-108379.2
aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg
AD-52666.1
A-108395.2
uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc
AD-52667.1
A-108317.2
aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg
AD-52668.1
A-108333.2
aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg
AD-52669.1
A-108349.2
uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg
AD-52670.1
A-108365.2
aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu
AD-52671.1
A-108381.2
uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu
AD-52672.1
A-108397.2
uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu
AD-52673.1
A-108319.2
aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg
AD-52674.1
A-108335.2
uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu
AD-52675.1
A-108351.2
uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa
AD-52676.1
A-108367.2
uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg
AD-52677.1
A-108383.2
uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa
AD-52678.1
A-108399.2
uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg
AD-52679.1
A-108321.2
aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa
AD-52680.1
A-108337.2
uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa
AD-52681.1
A-108353.2
aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa
AD-52682.1
A-108369.2
uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu
AD-52683.1
A-108385.2
aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa
AD-52684.1
A-108401.2
uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa
AD-52685.1
A-108417.2
aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg
AD-52686.1
A-108433.2
uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa
AD-52687.1
A-108449.2
uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu
AD-52688.1
A-108465.2
uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu
AD-52689.1
A-108481.2
aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg
AD-52690.1
A-108403.2
uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu
AD-52691.1
A-108419.2
uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa
AD-52692.1
A-108435.2
aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa
AD-52693.1
A-108451.2
aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc
AD-52694.1
A-108467.2
aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu
AD-52695.1
A-108483.2
uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu
AD-52696.1
A-108405.2
aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg
AD-52697.1
A-108421.2
uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa
AD-52698.1
A-108437.2
uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg
AD-52699.1
A-108453.2
aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu
AD-52700.1
A-108469.2
uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa
AD-52701.1
A-108485.2
uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu
AD-52702.1
A-108407.2
uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg
AD-52703.1
A-108423.2
uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu
AD-52704.1
A-108439.2
aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa
AD-52705.1
A-108455.2
uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu
AD-52706.1
A-108471.2
uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu
AD-52707.1
A-108487.2
uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu
AD-52708.1
A-108409.2
uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu
AD-52709.1
A-108425.2
uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg
AD-52710.1
A-108441.2
aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu
AD-52711.1
A-108457.2
uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc
AD-52712.1
A-108473.2
uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu
AD-52713.1
A-108489.2
aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu
AD-52714.1
A-108411.2
uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa
AD-52715.1
A-108427.2
uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa
AD-52716.1
A-108443.2
uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc
AD-52717.1
A-108459.2
uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg
AD-52718.1
A-108475.2
uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu
AD-52719.1
A-108491.2
aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu
AD-52720.1
A-108413.2
uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu
AD-52721.1
A-108429.2
uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc
AD-52722.1
A-108445.2
uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu
AD-52723.1
A-108461.2
aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu
AD-52724.1
A-108477.2
uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu
AD-52725.1
A-108493.2
aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu
AD-52726.1
A-108415.2
uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa
AD-52727.1
A-108431.2
uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu
AD-52728.1
A-108447.2
uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa
AD-52729.1
A-108463.2
uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc
AD-52730.1
A-108479.2
uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu
AD-52731.1
A-108495.2
aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa
AD-52732.1
A-108511.2
uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc
AD-52733.1
A-108527.2
aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa
AD-52734.1
A-108543.2
uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc
AD-52735.1
A-108559.2
aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc
AD-52736.1
A-108575.2
aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu
AD-52737.1
A-108497.2
aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg
AD-52739.1
A-108529.2
uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu
AD-52740.1
A-108545.2
uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc
AD-52741.1
A-108561.2
uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu
AD-52742.1
A-108577.2
uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa
AD-52743.1
A-108499.2
aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu
AD-52744.1
A-108515.2
uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu
AD-52745.1
A-108531.2
uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa
AD-52746.1
A-108547.2
uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu
AD-52747.1
A-108563.2
aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu
AD-52748.1
A-108579.2
aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa
AD-52749.1
A-108501.2
aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc
AD-52750.1
A-108517.2
aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa
AD-52751.1
A-108533.2
uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc
AD-52752.1
A-108549.2
uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa
AD-52753.1
A-108565.2
uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc
AD-52754.1
A-108581.2
uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc
AD-52755.1
A-108503.2
uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu
AD-52756.1
A-108519.2
uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc
AD-52757.1
A-108535.2
aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc
AD-52758.1
A-108551.2
aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu
AD-52759.1
A-108567.2
aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu
AD-52760.1
A-108583.2
aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu
AD-52761.1
A-108505.2
uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa
AD-52762.1
A-108521.2
aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg
AD-52763.1
A-108537.2
aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa
AD-52764.1
A-108553.2
aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg
AD-52765.1
A-108569.2
aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg
AD-52766.1
A-108585.2
uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg
AD-52767.1
A-108507.2
uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu
AD-52768.1
A-108523.2
aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu
AD-52769.1
A-108539.2
uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa
AD-52770.1
A-108555.2
uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa
AD-52771.1
A-108571.2
aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg
AD-52772.1
A-108587.2
aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu
AD-52773.1
A-108509.2
aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg
AD-52774.1
A-108525.2
uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu
AD-52775.1
A-108541.2
aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc
AD-52776.1
A-108557.2
uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc
AD-52777.1
A-108573.2
aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu
AD-52778.1
A-108589.2
aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa
AD-52779.1
A-108605.2
uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa
AD-52780.1
A-108621.2
uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu
AD-52781.1
A-108637.2
uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa
AD-52782.1
A-108653.2
uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa
AD-52783.1
A-108669.2
aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu
AD-52784.1
A-108591.2
aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc
AD-52785.1
A-108607.2
uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa
AD-52786.1
A-108623.2
uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu
AD-52787.1
A-108639.2
aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg
AD-52788.1
A-108655.2
uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc
AD-52789.1
A-108671.2
uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg
AD-52791.1
A-108609.2
uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg
AD-52792.1
A-108625.2
uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu
AD-52793.1
A-108641.2
aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa
AD-52794.1
A-108657.2
uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu
AD-52795.1
A-108673.2
uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu
AD-52796.1
A-108595.2
aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg
AD-52797.1
A-108611.2
uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa
AD-52798.1
A-108627.2
uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu
AD-52799.1
A-108643.2
uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa
AD-52800.1
A-108659.2
uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu
AD-52801.1
A-108675.2
aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc
AD-52802.1
A-108597.2
aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc
AD-52803.1
A-108613.2
uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc
AD-52804.1
A-108629.2
uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu
AD-52805.1
A-108645.2
uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa
AD-52806.1
A-108661.2
aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg
AD-52807.1
A-108677.2
uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu
AD-52808.1
A-108599.2
uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu
AD-52809.1
A-108615.2
aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu
AD-52810.1
A-108631.2
uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu
AD-52811.1
A-108647.2
uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc
AD-52812.1
A-108663.2
uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa
AD-52813.1
A-108601.2
aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg
AD-52814.1
A-108617.2
uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc
AD-52815.1
A-108633.2
uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa
AD-52816.1
A-108649.2
uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu
AD-52817.1
A-108665.2
uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc
AD-52818.1
A-108603.2
uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa
AD-52819.1
A-108619.2
aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu
AD-52820.1
A-108635.2
uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa
AD-52821.1
A-108651.2
uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu
AD-52822.1
A-108667.2
aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg
TABLE 11
Results of single dose screen using ANGPTL3 GalNac-conjugated dsRNA
Modified siRNAs were tested by transfection in Hep3b cells and by free-uptake
in primary cynomolgus monkey (PCH) cells at the above-stated doses.
500 nM
100 nM
10 nM
PCH
PCH
PCH
STDEV
STDEV
STDEV
STDEV
STDEV
10 nM
0.1 nM
Celsis
Celsis
Celsis
10 nM
0.1 nM
500 nM
100 nM
10 nM
DUPLEX ID
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
(RNAimax)
(RNAimax)
(FU)
(FU)
(FU)
AD1955/naïve FU
0.93
0.93
1.01
0.91
1.17
0.02
0.08
0.09
0.00
0.07
AD1955/naïve FU
1.02
1.09
1.07
1.07
0.92
0.06
0.04
0.02
0.00
0.03
AD1955/naïve FU
1.06
0.99
0.93
1.02
0.93
0.03
0.00
0.09
0.01
0.02
AD1955/naïve FU
1.05
0.90
1.05
1.03
1.03
0.04
0.02
0.01
0.05
0.01
AD1955/naïve FU
1.06
1.08
0.90
0.97
1.03
0.02
0.01
0.02
0.04
0.09
AD1955/naïve FU
0.90
1.03
1.05
1.00
0.94
0.04
0.03
0.01
0.04
0.05
AD-45165 (TTR)
0.91
0.98
1.06
0.98
0.96
0.05
0.01
0.05
0.00
0.00
AD-52953.1
0.06
0.34
0.15
0.17
0.46
0.00
0.01
0.00
0.01
0.01
AD-52954.1
0.09
0.39
0.17
0.20
0.55
0.00
0.01
0.00
0.01
0.00
AD-52955.1
0.11
0.59
0.38
0.41
0.75
0.01
0.04
0.02
0.01
0.12
AD-52956.1
0.31
0.94
0.79
0.94
1.17
0.01
0.00
0.02
0.06
0.02
AD-52957.1
0.13
0.61
0.35
0.38
0.73
0.01
0.00
0.01
0.00
0.04
AD-52958.1
0.19
0.74
0.66
0.71
0.97
0.01
0.01
0.02
0.07
0.06
AD-52960.1
0.14
0.59
0.31
0.32
0.55
0.01
0.01
0.00
0.02
0.02
AD-52961.1
0.05
0.66
0.27
0.24
0.49
0.00
0.00
0.00
0.02
0.02
AD-52962.1
0.83
0.89
1.03
1.02
1.26
0.02
0.05
0.07
0.07
0.07
AD-52963.1
0.07
0.72
0.46
0.56
0.91
0.00
0.00
0.00
0.00
0.06
AD-52964.1
0.13
0.73
0.41
0.47
0.68
0.01
0.03
0.02
0.03
0.01
AD-52965.1
0.07
0.44
0.16
0.18
0.43
0.00
0.01
0.00
0.01
0.01
AD-52966.1
0.12
0.76
0.67
0.72
0.96
0.00
0.02
0.05
0.01
0.01
AD-52967.1
0.10
0.75
0.44
0.58
0.89
0.01
0.04
0.02
0.03
0.04
AD-52968.1
1.01
0.96
0.87
0.91
1.15
0.00
0.01
0.09
0.03
0.02
AD-52969.1
0.04
0.46
0.22
0.29
0.59
0.00
0.00
0.01
0.02
0.04
AD-52970.1
0.06
0.45
0.27
0.30
0.51
0.00
0.00
0.01
0.02
0.00
AD-52971.1
0.08
0.55
0.20
0.22
0.45
0.00
0.00
0.01
0.02
0.05
AD-52972.1
0.10
0.73
0.41
0.49
0.81
0.00
0.01
0.01
0.02
0.01
AD-52973.1
0.11
0.73
0.36
0.46
0.75
0.01
0.01
0.03
0.02
0.02
AD-52974.1
1.00
0.95
1.00
1.09
1.27
0.01
0.01
0.08
0.05
0.06
AD-52975.1
0.07
0.54
0.25
0.34
0.66
0.00
0.01
0.01
0.01
0.03
AD-52976.1
0.17
0.59
0.35
0.41
0.65
0.00
0.02
0.04
0.01
0.01
AD-52977.1
0.07
0.45
0.16
0.25
0.50
0.01
0.02
0.00
0.02
0.03
AD-52978.1
0.10
0.72
0.39
0.53
0.77
0.00
0.02
0.00
0.08
0.03
AD-52979.1
0.54
0.92
0.99
1.12
1.28
0.01
0.02
0.02
0.04
0.05
AD-52980.1
0.29
0.85
0.67
0.85
1.03
0.01
0.01
0.05
0.05
0.04
AD-52981.1
0.07
0.44
0.20
0.26
0.59
0.01
0.02
0.00
0.00
0.03
AD-52982.1
0.28
0.87
0.67
0.99
1.14
0.01
0.01
0.04
0.00
0.01
AD-52983.1
0.06
0.40
0.14
0.40
0.46
0.00
0.00
0.01
0.05
0.02
AD-52984.1
0.29
0.87
0.66
0.74
1.09
0.01
0.02
0.01
0.00
0.00
AD-52985.1
0.72
0.87
0.89
1.18
1.22
0.03
0.00
0.05
0.03
0.16
AD-52986.1
0.08
0.47
0.24
0.30
0.48
0.00
0.02
0.02
0.00
0.06
AD-52987.1
0.16
0.83
0.42
0.73
1.09
0.00
0.00
0.01
0.02
0.02
AD-52988.1
0.11
0.73
0.42
0.60
0.96
0.01
0.04
0.00
0.00
0.10
AD-52989.1
0.05
0.48
0.15
0.42
0.46
0.00
0.02
0.00
0.02
0.00
AD-52990.1
0.14
0.86
0.33
0.45
0.77
0.00
0.01
0.00
0.02
0.05
AD-52991.1
0.16
0.86
0.58
0.69
1.05
0.00
0.00
0.02
0.00
0.02
AD-52992.1
0.08
0.65
0.42
0.56
0.90
0.00
0.01
0.02
0.01
0.00
AD-52993.1
0.13
0.87
0.53
0.76
1.08
0.02
0.03
0.04
0.04
0.00
AD-52994.1
0.10
0.52
0.28
0.33
0.53
0.01
0.00
0.02
0.00
0.01
AD-52995.1
0.06
0.56
0.19
0.41
0.60
0.00
0.01
0.04
0.02
0.05
AD-52996.1
0.09
0.68
0.26
0.47
0.68
0.00
0.03
0.01
0.04
0.01
AD-52997.1
0.59
1.03
0.87
0.51
1.25
0.05
0.01
0.00
0.01
0.01
AD-52998.1
0.09
0.79
0.44
0.55
0.85
0.00
0.00
0.04
0.03
0.10
AD-52999.1
0.08
0.57
0.17
0.36
0.84
0.01
0.00
0.01
0.02
0.00
AD-53000.1
0.38
0.94
0.58
0.67
0.85
0.01
0.02
0.03
0.03
0.02
AD-53001.1
0.05
0.48
0.21
0.18
0.40
0.00
0.00
0.01
0.00
0.05
AD-53002.1
0.07
0.65
0.43
0.48
0.80
0.00
0.05
0.04
0.01
0.02
AD-53003.1
0.05
0.46
0.31
0.34
0.56
0.01
0.01
0.00
0.02
0.05
AD-53004.1
0.05
0.36
0.29
0.66
0.57
0.00
0.01
0.03
0.35
0.02
AD-53005.1
0.05
0.72
0.32
0.58
0.83
0.01
0.00
0.01
0.29
0.00
AD-53006.1
0.21
0.82
0.66
0.77
1.03
0.01
0.00
0.02
0.07
0.02
AD-53007.1
0.12
0.76
0.55
0.73
0.74
0.01
0.00
0.00
0.08
0.20
AD-53008.1
0.07
0.68
0.28
0.36
0.84
0.00
0.02
0.01
0.05
0.03
AD-53009.1
0.10
0.61
0.48
0.60
0.91
0.00
0.02
0.01
0.01
0.06
AD-53010.1
0.05
0.58
0.47
0.54
0.84
0.00
0.02
0.00
0.02
0.03
AD-53011.1
0.07
0.65
0.29
0.34
0.84
0.00
0.03
0.07
0.01
0.04
AD-53012.1
0.06
0.55
0.36
0.45
0.70
0.00
0.03
0.02
0.02
0.00
AD-53013.1
0.11
0.85
0.59
0.70
1.01
0.00
0.00
0.03
0.03
0.02
AD-53014.1
0.16
0.78
0.61
0.78
1.11
0.00
0.02
0.01
0.05
0.00
AD-53015.1
0.03
0.35
0.25
0.37
0.46
0.01
0.01
0.01
0.00
0.01
AD-53016.1
0.03
0.56
0.40
0.58
1.01
0.00
0.01
0.02
0.06
0.09
AD-53017.1
0.07
0.71
0.64
0.78
0.98
0.00
0.01
0.01
0.05
0.00
AD-53018.1
0.30
0.96
0.75
0.97
1.14
0.00
0.02
0.02
0.03
0.05
AD-53019.1
0.27
0.99
0.77
1.05
1.31
0.00
0.01
0.01
0.04
0.00
AD-53020.1
0.04
0.64
0.32
0.45
0.69
0.00
0.00
0.03
0.02
0.03
AD-53021.1
0.04
0.68
0.36
0.48
0.70
0.01
0.01
0.02
0.07
0.00
AD-53022.1
0.05
0.76
0.36
0.59
1.04
0.01
0.01
0.02
0.03
0.06
AD-53023.1
0.10
0.83
0.69
0.84
0.97
0.01
0.01
0.06
0.02
0.01
AD-53024.1
0.09
0.44
0.23
0.23
0.44
0.00
0.00
0.03
0.01
0.02
AD-53025.1
0.09
0.87
0.58
0.80
1.09
0.00
0.03
0.01
0.04
0.04
AD-53026.1
0.05
0.60
0.35
0.46
0.77
0.01
0.01
0.02
0.05
0.03
AD-53027.1
0.02
0.32
0.26
0.30
0.45
0.00
0.01
0.02
0.03
0.02
AD-53028.1
0.19
0.82
0.77
0.95
1.04
0.01
0.04
0.05
0.01
0.03
AD-53029.1
0.02
0.52
0.32
0.41
0.72
0.00
0.00
0.01
0.02
0.07
AD-53030.1
0.09
0.42
0.15
0.16
0.46
0.00
0.00
0.00
0.00
0.02
AD-53031.1
0.12
0.79
0.63
0.73
1.04
0.02
0.05
0.02
0.04
0.03
AD-53032.1
0.12
0.71
0.41
0.59
0.90
0.01
0.00
0.02
0.04
0.00
AD-53033.1
0.02
0.48
0.20
0.21
0.51
0.00
0.02
0.02
0.01
0.00
AD-53034.1
0.04
0.52
0.31
0.36
0.71
0.00
0.01
0.07
0.02
0.01
AD-53035.1
0.02
0.63
0.34
0.50
0.85
0.00
0.02
0.03
0.00
0.03
AD-53036.1
0.10
0.57
0.31
0.35
0.65
0.01
0.01
0.03
0.03
0.01
AD-53037.1
0.08
0.47
0.27
0.36
0.60
0.00
0.02
0.01
0.03
0.01
AD-53038.1
0.05
0.85
0.48
0.63
1.08
0.00
0.05
0.00
0.02
0.05
AD-53039.1
0.08
0.82
0.45
0.64
0.97
0.00
0.01
0.01
0.03
0.00
AD-53040.1
0.05
0.79
0.46
0.62
0.97
0.01
0.01
0.01
0.05
0.06
AD-53041.1
0.06
0.72
0.59
0.61
0.86
0.00
0.01
0.05
0.06
0.03
AD-53042.1
0.08
0.85
0.30
0.35
0.81
0.01
0.00
0.00
0.03
0.03
AD-53043.1
0.63
1.00
0.92
1.04
1.07
0.03
0.00
0.06
0.03
0.07
AD-53044.1
0.05
0.91
0.35
0.61
0.97
0.01
0.01
0.01
0.04
0.02
AD-53045.1
0.20
1.00
0.85
1.00
0.98
0.00
0.03
0.04
0.01
0.04
AD-53046.1
0.07
0.70
0.44
0.62
1.12
0.00
0.01
0.03
0.00
0.09
AD-53059.1
0.35
1.04
0.75
0.85
0.86
0.01
0.01
0.03
0.02
0.04
AD-53060.1
0.34
0.85
0.72
0.96
0.82
0.00
0.01
0.02
0.01
0.02
AD-53061.1
0.17
0.94
0.36
0.37
0.59
0.00
0.00
0.02
0.00
0.02
AD-53062.1
0.09
0.76
0.43
0.47
0.69
0.01
0.01
0.01
0.03
0.01
AD-53063.1
0.06
0.48
0.18
0.16
0.25
0.00
0.01
0.01
0.01
0.02
AD-53064.1
0.07
0.59
0.22
0.22
0.48
0.01
0.02
0.01
0.02
0.06
AD-53065.1
0.08
0.97
0.45
0.39
0.64
0.01
0.01
0.02
0.01
0.01
AD-53066.1
0.12
0.99
0.73
0.67
0.88
0.01
0.03
0.01
0.01
0.05
AD-53067.1
0.12
1.08
0.59
0.60
0.79
0.00
0.12
0.01
0.01
0.03
AD-53068.1
0.09
0.98
0.46
0.59
0.83
0.00
0.03
0.04
0.07
0.05
AD-53069.1
0.04
0.69
0.35
0.43
0.59
0.00
0.01
0.01
0.04
0.01
AD-53070.1
0.17
1.12
0.88
0.83
0.98
0.00
0.01
0.04
0.00
0.01
AD-53071.1
0.07
0.70
0.23
0.23
0.43
0.00
0.00
0.02
0.00
0.01
AD-53072.1
0.10
0.90
0.49
0.48
0.75
0.01
0.05
0.00
0.01
0.02
AD-53073.1
0.07
0.63
0.27
0.30
0.43
0.00
0.00
0.01
0.01
0.00
AD-53074.1
0.07
0.88
0.46
0.49
0.62
0.01
0.08
0.01
0.06
0.03
AD-53075.1
0.05
0.76
0.29
0.35
0.50
0.01
0.01
0.00
0.02
0.03
AD-53076.1
0.09
0.80
0.31
0.40
0.54
0.01
0.01
0.02
0.05
0.02
AD-53077.1
0.07
0.96
0.29
0.28
0.49
0.00
0.03
0.00
0.01
0.01
AD-53078.1
0.16
0.95
0.51
0.51
0.70
0.00
0.04
0.01
0.01
0.06
AD-53079.1
0.08
0.96
0.59
0.67
0.83
0.00
0.02
0.01
0.03
0.01
AD-53080.1
0.04
0.63
0.20
0.22
0.43
0.00
0.01
0.00
0.01
0.01
AD-53081.1
0.16
1.02
0.63
0.75
0.87
0.00
0.09
0.00
0.02
0.05
AD-53082.1
0.06
0.94
0.50
0.52
0.66
0.01
0.06
0.02
0.03
0.03
AD-53083.1
0.14
0.87
0.48
0.50
0.80
0.01
0.02
0.04
0.06
0.01
AD-53084.1
0.12
0.95
0.50
0.47
0.72
0.01
0.03
0.04
0.00
0.00
AD-53085.1
0.27
1.02
0.68
0.81
0.99
0.01
0.01
0.01
0.05
0.02
AD-53086.1
0.05
0.60
0.26
0.25
0.48
0.00
0.01
0.03
0.00
0.01
AD-53087.1
0.05
0.56
0.32
0.39
0.53
0.00
0.01
0.01
0.03
0.02
AD-53088.1
0.09
0.89
0.53
0.69
0.87
0.00
0.01
0.02
0.04
0.02
AD-53089.1
0.29
0.97
0.58
0.57
0.78
0.01
0.00
0.02
0.02
0.02
AD-53090.1
0.13
0.86
0.56
0.55
0.73
0.00
0.01
0.01
0.03
0.00
AD-53091.1
0.12
0.82
0.27
0.35
0.66
0.00
0.03
0.03
0.01
0.07
AD-53092.1
0.05
0.66
0.26
0.29
0.42
0.00
0.01
0.02
0.04
0.02
AD-53093.1
0.08
0.68
0.36
0.44
0.55
0.00
0.02
0.03
0.04
0.10
AD-53094.1
0.32
1.00
1.05
0.92
1.11
0.02
0.01
0.01
0.00
0.03
AD-53095.1
0.14
0.77
0.29
0.29
0.49
0.00
0.02
0.00
0.01
0.01
AD-53096.1
0.30
0.96
0.61
0.57
0.73
0.03
0.01
0.02
0.02
0.01
AD-53097.1
0.37
0.97
0.67
0.82
0.86
0.01
0.01
0.01
0.02
0.01
AD-53098.1
0.06
0.65
0.22
0.30
0.43
0.00
0.03
0.03
0.00
0.01
AD-53099.1
0.34
0.99
0.61
0.81
0.91
0.00
0.00
0.04
0.02
0.06
AD-53100.1
0.31
1.04
0.95
1.03
1.00
0.02
0.01
0.06
0.02
0.17
AD-53101.1
0.46
0.93
0.63
0.69
0.78
0.00
0.01
0.04
0.03
0.04
AD-53102.1
0.23
0.80
0.60
0.55
0.66
0.00
0.03
0.01
0.02
0.03
AD-53103.1
0.05
0.61
0.27
0.32
0.50
0.01
0.02
0.00
0.01
0.00
AD-53104.1
0.13
0.80
0.64
0.68
0.77
0.00
0.02
0.03
0.01
0.05
AD-53105.1
0.15
0.77
0.43
0.65
0.77
0.01
0.03
0.02
0.02
0.05
AD-53106.1
0.16
0.87
0.72
0.70
0.83
0.01
0.02
0.00
0.00
0.04
AD-53107.1
0.19
0.95
0.62
0.65
0.90
0.00
0.02
0.01
0.03
0.04
AD-53108.1
0.22
0.94
0.60
0.68
0.81
0.00
0.01
0.00
0.03
0.04
AD-53109.1
0.16
1.01
0.82
0.78
0.96
0.01
0.08
0.04
0.01
0.07
AD-53110.1
0.10
0.86
0.79
0.77
0.94
0.00
0.05
0.03
0.01
0.05
AD-53111.1
0.22
0.78
0.94
0.85
1.04
0.01
0.01
0.01
0.01
0.07
AD-53112.1
0.09
0.96
0.64
0.65
0.86
0.01
0.02
0.07
0.07
0.00
AD-53113.1
0.10
0.97
0.71
0.77
0.88
0.01
0.05
0.01
0.02
0.01
AD-53114.1
0.19
0.83
0.48
0.52
0.66
0.01
0.01
0.02
0.01
0.00
AD-53115.1
0.10
0.59
0.42
0.44
0.66
0.01
0.03
0.04
0.00
0.02
AD-53116.1
0.11
0.87
0.82
0.85
0.95
0.00
0.05
0.05
0.05
0.05
AD-53117.1
0.52
0.64
1.21
1.00
1.08
0.01
0.03
0.09
0.04
0.07
AD-53118.1
0.19
1.04
0.60
0.72
0.94
0.00
0.07
0.02
0.05
0.06
AD-53119.1
0.06
0.77
0.44
0.47
0.64
0.01
0.03
0.00
0.01
0.01
AD-53120.1
0.10
0.97
0.78
0.89
1.01
0.01
0.04
0.05
0.01
0.04
AD-53121.1
0.23
0.80
0.58
0.69
0.90
0.01
0.02
0.04
0.02
0.06
AD-53122.1
0.09
0.80
0.90
0.94
1.09
0.01
0.07
0.02
0.04
0.10
AD-53123.1
0.27
0.74
0.95
0.93
0.97
0.00
0.01
0.03
0.01
0.08
AD-53124.1
0.08
0.81
0.33
0.34
0.61
0.01
0.02
0.00
0.01
0.01
AD-53125.1
0.08
0.82
0.34
0.38
0.58
0.00
0.02
0.00
0.01
0.07
AD-53126.1
0.15
0.95
0.70
0.86
1.06
0.01
0.04
0.05
0.02
0.00
AD-53127.1
0.21
0.81
0.62
0.75
0.91
0.02
0.04
0.01
0.03
0.00
AD-53128.1
0.08
0.79
0.80
1.14
1.09
0.00
0.06
0.04
0.01
0.03
AD-53129.1
0.48
0.78
1.05
1.00
1.10
0.00
0.01
0.06
0.01
0.03
AD-53130.1
0.25
1.08
0.63
0.72
0.88
0.01
0.02
0.00
0.01
0.00
AD-53131.1
0.14
0.96
0.54
0.57
0.81
0.02
0.02
0.05
0.01
0.04
AD-53132.1
0.03
0.54
0.24
0.27
0.49
0.00
0.02
0.02
0.00
0.01
AD-53133.1
0.12
0.76
0.50
0.67
0.93
0.00
0.03
0.01
0.01
0.06
AD-53134.1
0.28
0.86
1.14
0.81
0.97
0.01
0.04
0.05
0.02
0.04
AD-53135.1
0.47
0.74
1.03
0.94
1.09
0.01
0.03
0.04
0.07
0.04
AD-53136.1
0.09
0.99
0.64
0.69
0.94
0.01
0.05
0.01
0.05
0.02
AD-53137.1
0.08
0.75
0.39
0.39
0.59
0.01
0.03
0.00
0.00
0.00
AD-53138.1
0.04
0.71
0.33
0.34
0.60
0.00
0.02
0.00
0.03
0.00
AD-53139.1
0.11
0.76
0.55
0.66
0.84
0.01
0.01
0.06
0.01
0.02
AD-53140.1
0.09
0.71
0.64
0.71
0.86
0.00
0.04
0.01
0.02
0.02
AD-53141.1
0.24
1.09
0.77
0.91
0.93
0.00
0.01
0.00
0.06
0.00
AD-53142.1
0.13
0.95
0.55
0.70
0.82
0.01
0.03
0.03
0.04
0.02
AD-53143.1
0.13
0.91
0.67
0.83
0.94
0.01
0.00
0.03
0.03
0.07
AD-53144.1
0.10
0.72
0.54
0.69
0.84
0.01
0.03
0.01
0.03
0.00
AD-53145.1
0.08
0.72
0.70
0.78
0.88
0.01
0.03
0.01
0.08
0.02
AD-53146.1
0.83
1.07
0.85
0.96
0.98
0.01
0.06
0.00
0.05
0.00
AD-53147.1
0.08
0.56
0.27
0.34
0.47
0.00
0.01
0.01
0.01
0.01
AD-53148.1
0.06
0.81
0.61
0.68
0.74
0.01
0.00
0.03
0.06
0.05
AD-53149.1
0.23
0.86
0.71
0.83
0.92
0.01
0.02
0.06
0.02
0.03
AD-53150.1
0.41
0.70
1.03
1.09
1.03
0.03
0.06
0.03
0.04
0.01
TABLE 12
Dose response screen results for ANGPTL3 GalNac-conjugated
dsRNA sequences
A subset of active siRNAs from the single dose
screen (refer to data in Table 11) was tested in a dose
response experiment by free uptake in PCH cells. A subset
of these active siRNAs was also tested in dose response in
Hep3B cells by transfection.
IC50 (nM)
Free uptake
Transfection (RNAiMax)
AD-53063.1
1.60
0.03
AD-53001.1
2.27
0.01
AD-53015.1
2.90
0.02
AD-52953.1
2.94
0.03
AD-52986.1
3.30
0.03
AD-53024.1
3.42
0.02
AD-53033.1
3.42
0.02
AD-53027.1
3.84
0.01
AD-53030.1
3.90
0.03
AD-53080.1
4.08
0.04
AD-53073.1
4.20
0.05
AD-52965.1
4.63
ND
AD-53092.1
5.37
ND
AD-53132.1
5.54
ND
AD-52983.1
5.55
ND
AD-52954.1
5.67
ND
AD-52961.1
6.37
ND
AD-52994.1
6.43
ND
AD-53098.1
6.58
ND
AD-52970.1
6.71
ND
AD-53075.1
6.74
ND
AD-53086.1
7.08
ND
AD-52971.1
7.50
ND
AD-53064.1
8.33
ND
AD-53147.1
8.34
ND
AD-52969.1
8.86
ND
AD-53077.1
8.98
ND
AD-52981.1
9.44
ND
AD-52977.1
10.45
ND
AD-53071.1
11.19
ND
AD-52960.1
13.03
ND
AD-53095.1
21.31
ND
AD-53103.1
21.92
ND
TABLE 13
Results of single dose screen using sequences listed in Table 10.
STDEV
STDEV
STDEV
Duplex
10 nM
0.1 nM
0.025 nM
10 nM
0.1 nM
0.025 nM
AD-52719.1
0.01
0.60
0.35
0.000
0.093
0.002
AD-52717.1
0.02
0.31
0.32
0.001
0.014
0.008
AD-52713.1
0.02
0.37
0.36
0.001
0.011
0.007
AD-52711.1
0.03
0.22
0.23
0.005
0.011
0.009
AD-52718.1
0.03
0.31
0.39
0.000
0.025
0.023
AD-52687.1
0.03
0.37
0.38
0.005
0.020
0.002
AD-52699.1
0.03
0.25
0.21
0.002
0.011
0.002
AD-52679.1
0.03
0.51
0.24
0.345
0.008
AD-52689.1
0.03
0.44
0.42
0.000
0.039
0.002
AD-52700.1
0.03
0.56
0.57
0.005
0.044
0.020
AD-52637.1
0.04
0.27
0.23
0.001
0.003
0.005
AD-52730.1
0.04
0.61
0.59
0.005
0.053
0.014
AD-52725.1
0.04
0.62
0.61
0.002
0.027
0.012
AD-52688.1
0.04
0.23
0.20
0.006
0.012
0.011
AD-52661.1
0.04
0.61
0.25
0.001
0.449
0.009
AD-52667.1
0.04
0.28
0.22
0.004
0.018
0.013
AD-52665.1
0.04
0.43
0.48
0.007
0.019
0.009
AD-52638.1
0.04
0.28
0.25
0.000
0.016
0.027
AD-52724.1
0.05
0.86
0.76
0.001
0.055
0.011
AD-52705.1
0.05
0.74
0.65
0.004
0.022
0.016
AD-52708.1
0.05
0.53
0.52
0.001
0.034
0.013
AD-52659.1
0.05
0.56
0.48
0.000
0.000
0.033
AD-52678.1
0.05
0.53
0.53
0.002
0.034
0.000
AD-52670.1
0.05
0.35
0.33
0.002
0.009
0.003
AD-52695.1
0.05
0.63
0.67
0.001
0.012
0.013
AD-52704.1
0.05
0.55
0.53
0.002
0.005
0.034
AD-52683.1
0.05
0.36
0.28
0.002
0.021
0.011
AD-52673.1
0.05
0.22
0.19
0.023
0.010
0.002
AD-52721.1
0.05
0.60
0.53
0.003
0.006
0.029
AD-52710.1
0.05
0.56
0.40
0.007
0.073
0.000
AD-52714.1
0.05
0.40
0.51
0.000
0.016
0.003
AD-52686.1
0.05
0.57
0.60
0.003
0.014
0.000
AD-52645.1
0.05
0.62
0.59
0.004
0.030
0.003
AD-52662.1
0.05
0.55
0.52
0.002
0.030
0.008
AD-52720.1
0.05
0.50
0.46
0.003
0.007
0.011
AD-52654.1
0.05
0.29
0.36
0.008
0.037
0.014
AD-52680.1
0.06
0.48
0.41
0.001
0.019
0.026
AD-52723.1
0.06
0.84
0.76
0.001
0.041
0.004
AD-52726.1
0.06
0.72
0.66
0.003
0.028
0.016
AD-52701.1
0.06
0.67
0.39
0.001
0.003
0.002
AD-52694.1
0.06
0.68
0.59
0.004
0.040
0.012
AD-52685.1
0.06
0.30
0.25
0.002
0.013
0.016
AD-52728.1
0.06
0.80
0.79
0.005
0.043
0.015
AD-52676.1
0.06
0.68
0.67
0.002
0.023
0.029
AD-52639.1
0.06
0.47
0.45
0.000
0.005
0.007
AD-52722.1
0.06
0.81
0.93
0.005
0.004
0.027
AD-52682.1
0.06
0.87
0.73
0.009
0.038
0.014
AD-52660.1
0.07
0.69
0.68
0.002
0.014
0.017
AD-52709.1
0.07
0.89
0.82
0.001
0.013
0.020
AD-52643.1
0.07
0.27
0.24
0.006
0.016
0.012
AD-52696.1
0.07
0.53
0.46
0.003
0.026
0.007
AD-52657.1
0.08
0.60
0.58
0.008
0.030
0.006
AD-52706.1
0.08
0.84
0.78
0.001
0.021
0.019
AD-52653.1
0.08
0.41
0.45
0.057
0.004
0.029
AD-52656.1
0.08
0.65
0.50
0.004
0.022
0.012
AD-52693.1
0.09
0.61
0.62
0.007
0.021
0.018
AD-52692.1
0.09
0.54
0.52
0.023
0.018
0.033
AD-52674.1
0.10
0.79
0.64
0.001
0.008
0.028
AD-52648.1
0.10
0.67
0.53
0.002
0.013
0.028
AD-52651.1
0.10
0.84
0.73
0.000
0.000
0.007
AD-52641.1
0.10
0.62
0.50
0.004
0.172
0.002
AD-52707.1
0.10
0.92
0.81
0.001
0.018
0.032
AD-52671.1
0.11
0.87
0.84
0.005
0.034
0.025
AD-52650.1
0.12
0.88
0.94
0.007
0.013
0.041
AD-52642.1
0.12
0.90
0.76
0.015
0.022
0.004
AD-52675.1
0.13
0.94
0.89
0.001
0.018
0.044
AD-52647.1
0.13
0.80
0.79
0.031
0.008
0.023
AD-52716.1
0.14
0.61
0.69
0.010
0.060
0.013
AD-52649.1
0.14
0.31
0.29
0.136
0.020
0.006
AD-52677.1
0.16
1.01
0.72
0.059
0.040
0.007
AD-52697.1
0.16
0.86
0.77
0.012
0.021
0.015
AD-52715.1
0.17
0.90
0.89
0.005
0.009
0.022
AD-52691.1
0.18
0.93
0.88
0.004
0.036
0.017
AD-52698.1
0.20
0.97
0.87
0.010
0.028
0.000
AD-52672.1
0.20
0.70
0.66
0.170
0.014
0.019
AD-52712.1
0.29
0.92
0.90
0.007
0.036
0.004
AD-52690.1
0.30
0.95
0.85
0.115
0.032
0.004
AD-52640.1
0.30
1.04
0.91
0.018
0.046
0.013
AD-52684.1
0.31
0.90
0.94
0.014
0.018
0.014
AD-52666.1
0.32
1.04
0.91
0.013
0.005
0.004
AD-52703.1
0.32
1.02
0.96
0.016
0.015
0.005
AD-52729.1
0.33
1.02
0.87
0.032
0.020
0.008
AD-52668.1
0.35
0.94
0.90
0.029
0.046
0.026
AD-52681.1
0.57
1.00
0.99
0.003
0.034
0.039
AD-52702.1
0.72
1.02
0.92
0.658
0.060
0.014
AD-52727.1
0.73
1.03
0.91
0.004
0.065
0.027
AD-52663.1
0.78
1.05
0.96
0.027
0.010
0.005
AD-52669.1
0.91
0.91
0.94
0.004
0.049
0.032
AD-1955
0.95
0.84
0.95
0.005
0.021
0.019
AD-1955
0.97
1.07
1.03
0.000
0.021
0.015
AD-1955
1.01
1.08
1.01
0.035
0.011
0.005
mock
1.02
0.96
0.97
0.030
0.037
0.005
AD-1955
1.08
1.03
1.02
0.032
0.051
0.005
AD-52652.1
1.13
1.11
1.02
0.028
0.043
0.020
AD-52658.1
1.33
1.10
0.93
0.091
0.043
0.018
AD-52664.1
1.49
0.95
0.88
0.438
0.019
0.009
AD-52752.1
0.03
0.43
0.69
0.002
0.015
0.017
AD-52741.1
0.03
0.56
0.86
0.001
0.044
0.021
AD-52804.1
0.03
0.49
0.89
0.001
0.002
0.017
AD-52764.1
0.03
0.54
0.79
0.005
0.016
0.078
AD-52770.1
0.03
0.58
0.78
0.000
0.006
0.027
AD-52735.1
0.03
0.31
0.46
0.003
0.031
0.009
AD-52810.1
0.03
0.67
0.86
0.001
0.013
0.025
AD-52759.1
0.03
0.54
0.79
0.000
0.018
0.023
AD-52736.1
0.03
0.51
0.60
0.004
0.012
0.023
AD-52775.1
0.03
0.54
0.73
0.005
0.024
0.022
AD-52758.1
0.03
0.57
0.78
0.001
0.014
0.050
AD-52743.1
0.03
0.45
0.67
0.002
0.018
0.033
AD-52747.1
0.04
0.57
0.84
0.002
0.061
0.058
AD-52819.1
0.04
0.26
0.45
0.005
0.001
0.022
AD-52765.1
0.04
0.68
0.83
0.000
0.013
0.053
AD-52754.1
0.04
0.76
1.00
0.000
0.007
0.015
AD-52787.1
0.05
0.55
0.68
0.001
0.043
0.060
AD-52791.1
0.05
0.70
0.91
0.001
0.014
0.084
AD-52811.1
0.05
0.73
0.84
0.002
0.014
0.058
AD-52817.1
0.05
0.77
0.92
0.003
0.011
0.031
AD-52745.1
0.06
0.62
0.77
0.007
0.021
0.000
AD-52749.1
0.06
0.63
0.88
0.005
0.037
0.043
AD-52740.1
0.06
0.83
0.94
0.007
0.012
0.051
AD-52796.1
0.06
0.72
0.92
0.003
0.021
0.054
AD-52820.1
0.06
0.90
0.87
0.001
0.026
0.064
AD-52809.1
0.06
0.76
0.90
0.001
0.037
0.027
AD-52760.1
0.06
0.81
0.97
0.001
0.056
0.047
AD-52767.1
0.07
0.55
0.55
0.001
0.016
0.013
AD-52734.1
0.07
0.61
0.64
0.004
0.003
0.003
AD-52794.1
0.07
0.94
0.87
0.007
0.014
0.051
AD-52797.1
0.07
0.69
0.87
0.004
0.000
0.038
AD-52737.1
0.08
0.70
0.84
0.004
0.031
0.012
AD-52812.1
0.08
0.75
0.88
0.004
0.000
0.056
AD-52748.1
0.08
0.70
0.89
0.001
0.010
0.009
AD-52782.1
0.08
0.68
0.78
0.004
0.023
0.011
AD-52816.1
0.08
0.71
0.88
0.003
0.042
0.060
AD-52763.1
0.08
0.68
0.77
0.002
0.013
0.026
AD-52788.1
0.08
0.89
1.00
0.004
0.017
0.034
AD-52762.1
0.08
0.78
0.91
0.007
0.046
0.009
AD-52785.1
0.08
0.88
0.95
0.002
0.004
0.019
AD-52800.1
0.09
0.82
0.94
0.001
0.040
0.005
AD-52792.1
0.09
0.93
0.94
0.002
0.018
0.037
AD-52784.1
0.10
0.84
0.92
0.000
0.066
0.032
AD-52746.1
0.10
0.82
0.93
0.002
0.060
0.059
AD-52814.1
0.10
0.85
0.88
0.002
0.042
0.013
AD-52751.1
0.10
0.88
0.98
0.005
0.030
0.067
AD-52786.1
0.10
0.81
0.81
0.006
0.028
0.048
AD-52755.1
0.10
0.93
0.99
0.003
0.032
0.048
AD-52808.1
0.11
0.98
0.92
0.000
0.038
0.032
AD-52815.1
0.11
0.96
0.96
0.002
0.009
0.000
AD-52805.1
0.11
0.79
0.86
0.003
0.050
0.008
AD-52777.1
0.11
0.88
0.94
0.001
0.065
0.000
AD-52756.1
0.11
0.92
0.91
0.003
0.032
0.004
AD-52733.1
0.12
0.66
0.65
0.005
0.071
0.022
AD-52739.1
0.13
0.83
0.95
0.002
0.008
0.061
AD-52780.1
0.13
0.70
0.67
0.012
0.021
0.059
AD-52798.1
0.13
0.64
0.97
0.001
0.006
0.038
AD-52776.1
0.14
0.97
0.94
0.011
0.029
0.023
AD-52753.1
0.15
0.88
1.09
0.001
0.048
0.005
AD-52778.1
0.16
0.76
0.69
0.003
0.067
0.003
AD-52744.1
0.16
0.90
0.91
0.002
0.000
0.049
AD-52750.1
0.16
0.87
1.01
0.000
0.060
0.055
AD-52774.1
0.17
0.71
0.89
0.002
0.010
0.017
AD-52803.1
0.18
0.87
0.92
0.015
0.026
0.040
AD-52821.1
0.18
0.86
0.87
0.005
0.046
0.055
AD-52781.1
0.18
0.78
0.66
0.008
0.000
0.023
AD-52779.1
0.20
0.83
0.66
0.002
0.024
0.016
AD-52793.1
0.20
0.74
0.88
0.010
0.025
0.069
AD-52799.1
0.20
0.75
1.01
0.005
0.018
0.010
AD-52761.1
0.22
0.83
0.92
0.000
0.024
0.023
AD-52768.1
0.22
0.96
0.97
0.001
ND
0.028
AD-52757.1
0.23
1.02
0.95
0.018
0.040
0.042
AD-52806.1
0.24
0.96
0.87
0.011
0.084
0.055
AD-52771.1
0.25
0.92
0.98
0.010
0.018
0.048
AD-52802.1
0.30
0.95
1.00
0.010
0.019
0.005
AD-52731.1
0.30
0.85
0.75
0.001
0.067
0.022
AD-52813.1
0.30
1.07
0.98
0.001
0.109
0.014
AD-52742.1
0.31
0.95
1.03
0.005
0.028
0.056
AD-52766.1
0.35
0.97
1.00
0.010
0.024
0.044
AD-52732.1
0.41
0.79
0.73
0.004
0.016
0.039
AD-52773.1
0.43
0.99
0.92
0.004
0.029
0.022
AD-52772.1
0.43
1.00
1.02
0.006
0.000
0.065
AD-52822.1
0.44
0.68
0.81
0.004
0.010
0.016
AD-52783.1
0.45
0.66
0.76
0.009
0.036
0.019
AD-52789.1
0.50
0.68
0.78
0.010
0.053
0.004
AD-52795.1
0.50
0.82
0.69
0.000
0.080
0.054
AD-52801.1
0.54
0.70
0.79
0.018
0.038
0.035
AD-52807.1
0.57
0.76
0.93
0.006
0.011
0.032
AD-52769.1
0.76
0.97
0.92
0.015
0.085
0.045
AD-1955
0.90
0.96
1.04
0.018
0.165
0.010
AD-52818.1
0.92
1.03
0.92
0.009
0.010
0.063
AD-1955
1.01
0.90
0.96
0.005
0.031
0.019
AD-1955
1.05
1.09
1.00
0.046
0.085
0.005
AD-1955
1.05
1.07
1.00
0.010
0.031
0.039
mock
1.20
0.98
0.92
0.000
0.014
0.005
mock
1.25
0.99
1.00
0.006
0.005
0.034
TABLE 14
Results of a dose response screen using a subset of sequences
from Table 13.
A subset of active ANGPTL3 siRNAs from Table 10 were
tested by transfection in Hep3B cells in dose response screens.
Duplex
IC50 (nM)
AD-52819.1
0.0036
AD-52667.1
0.0037
AD-52638.1
0.0048
AD-52673.1
0.0049
AD-52711.1
0.0050
AD-52661.1
0.0054
AD-52654.1
0.0058
AD-52637.1
0.0058
AD-52643.1
0.0060
AD-52685.1
0.0062
AD-52670.1
0.0064
AD-52679.1
0.0064
AD-52649.1
0.0066
AD-52683.1
0.0069
AD-52688.1
0.0071
AD-52717.1
0.0072
AD-52699.1
0.0073
AD-52714.1
0.0086
AD-52718.1
0.0088
AD-52735.1
0.0093
AD-52653.1
0.0102
AD-52687.1
0.0109
AD-52680.1
0.0120
AD-52713.1
0.0133
AD-52720.1
0.0143
AD-52639.1
0.0161
AD-52696.1
0.0163
AD-52662.1
0.0179
AD-52659.1
0.0180
AD-52710.1
0.0195
AD-52689.1
0.0216
AD-52787.1
0.0242
AD-52765.1
0.0318
TABLE 15
IDs of duplex pairs for which both an unconjuaged and a GalNac-
conjugated version were synthesized and tested
These duplexes have the same sequence and modification pattern.
Unconjugated duplex ID
GalNac conjugated duplex ID
AD-52637.1
AD-52953.1
AD-52638.1
AD-52954.1
AD-52639.1
AD-52955.1
AD-52640.1
AD-52956.1
AD-52641.1
AD-52957.1
AD-52642.1
AD-52958.1
AD-52643.1
None
None
AD-52960.1
None
AD-52961.1
AD-52645.1
AD-52962.1
AD-52647.1
AD-52963.1
AD-52648.1
AD-52964.1
AD-52649.1
AD-52965.1
AD-52650.1
AD-52966.1
AD-52651.1
AD-52967.1
AD-52652.1
AD-52968.1
AD-52653.1
AD-52969.1
AD-52654.1
AD-52970.1
None
AD-52971.1
AD-52656.1
AD-52972.1
AD-52657.1
AD-52973.1
AD-52658.1
AD-52974.1
AD-52659.1
AD-52975.1
AD-52660.1
AD-52976.1
AD-52661.1
AD-52977.1
AD-52662.1
AD-52978.1
AD-52663.1
AD-52979.1
AD-52664.1
AD-52980.1
AD-52665.1
AD-52981.1
AD-52666.1
AD-52982.1
AD-52667.1
AD-52983.1
AD-52668.1
AD-52984.1
AD-52669.1
AD-52985.1
AD-52670.1
AD-52986.1
AD-52671.1
AD-52987.1
AD-52672.1
AD-52988.1
AD-52673.1
AD-52989.1
AD-52674.1
AD-52990.1
AD-52675.1
AD-52991.1
AD-52676.1
AD-52992.1
AD-52677.1
AD-52993.1
AD-52678.1
AD-52994.1
AD-52679.1
AD-52995.1
AD-52680.1
AD-52996.1
AD-52681.1
AD-52997.1
AD-52682.1
AD-52998.1
AD-52683.1
AD-52999.1
AD-52684.1
AD-53000.1
AD-52685.1
AD-53001.1
AD-52686.1
AD-53002.1
AD-52687.1
AD-53003.1
AD-52688.1
AD-53004.1
AD-52689.1
AD-53005.1
AD-52690.1
AD-53006.1
AD-52691.1
AD-53007.1
AD-52692.1
AD-53008.1
AD-52693.1
AD-53009.1
AD-52694.1
AD-53010.1
AD-52695.1
AD-53011.1
AD-52696.1
AD-53012.1
AD-52697.1
AD-53013.1
AD-52698.1
AD-53014.1
AD-52699.1
AD-53015.1
AD-52700.1
AD-53016.1
AD-52701.1
AD-53017.1
AD-52702.1
AD-53018.1
AD-52703.1
AD-53019.1
AD-52704.1
AD-53020.1
AD-52705.1
AD-53021.1
AD-52706.1
AD-53022.1
AD-52707.1
AD-53023.1
AD-52708.1
AD-53024.1
AD-52709.1
AD-53025.1
AD-52710.1
AD-53026.1
AD-52711.1
AD-53027.1
AD-52712.1
AD-53028.1
AD-52713.1
AD-53029.1
AD-52714.1
AD-53030.1
AD-52715.1
AD-53031.1
AD-52716.1
AD-53032.1
AD-52717.1
AD-53033.1
AD-52718.1
AD-53034.1
AD-52719.1
AD-53035.1
AD-52720.1
AD-53036.1
AD-52721.1
AD-53037.1
AD-52722.1
AD-53038.1
AD-52723.1
AD-53039.1
AD-52724.1
AD-53040.1
AD-52725.1
AD-53041.1
AD-52726.1
AD-53042.1
AD-52727.1
AD-53043.1
AD-52728.1
AD-53044.1
AD-52729.1
AD-53045.1
AD-52730.1
AD-53046.1
AD-52731.1
AD-53059.1
AD-52732.1
AD-53060.1
AD-52733.1
AD-53061.1
AD-52734.1
AD-53062.1
AD-52735.1
AD-53063.1
AD-52736.1
AD-53064.1
AD-52737.1
AD-53065.1
None
AD-53066.1
AD-52739.1
AD-53067.1
AD-52740.1
AD-53068.1
AD-52741.1
AD-53069.1
AD-52742.1
AD-53070.1
AD-52743.1
AD-53071.1
AD-52744.1
AD-53072.1
AD-52745.1
AD-53073.1
AD-52746.1
AD-53074.1
AD-52747.1
AD-53075.1
AD-52748.1
AD-53076.1
AD-52749.1
AD-53077.1
AD-52750.1
AD-53078.1
AD-52751.1
AD-53079.1
AD-52752.1
AD-53080.1
AD-52753.1
AD-53081.1
AD-52754.1
AD-53082.1
AD-52755.1
AD-53083.1
AD-52756.1
AD-53084.1
AD-52757.1
AD-53085.1
AD-52758.1
AD-53086.1
AD-52759.1
AD-53087.1
AD-52760.1
AD-53088.1
AD-52761.1
AD-53089.1
AD-52762.1
AD-53090.1
AD-52763.1
AD-53091.1
AD-52764.1
AD-53092.1
AD-52765.1
AD-53093.1
AD-52766.1
AD-53094.1
AD-52767.1
AD-53095.1
AD-52768.1
AD-53096.1
AD-52769.1
AD-53097.1
AD-52770.1
AD-53098.1
AD-52771.1
AD-53099.1
AD-52772.1
AD-53100.1
AD-52773.1
AD-53101.1
AD-52774.1
AD-53102.1
AD-52775.1
AD-53103.1
AD-52776.1
AD-53104.1
AD-52777.1
AD-53105.1
AD-52778.1
AD-53106.1
AD-52779.1
AD-53107.1
AD-52780.1
AD-53108.1
AD-52781.1
AD-53109.1
AD-52782.1
AD-53110.1
AD-52783.1
AD-53111.1
AD-52784.1
AD-53112.1
AD-52785.1
AD-53113.1
AD-52786.1
AD-53114.1
AD-52787.1
AD-53115.1
AD-52788.1
AD-53116.1
AD-52789.1
AD-53117.1
None
AD-53118.1
AD-52791.1
AD-53119.1
AD-52792.1
AD-53120.1
AD-52793.1
AD-53121.1
AD-52794.1
AD-53122.1
AD-52795.1
AD-53123.1
AD-52796.1
AD-53124.1
AD-52797.1
AD-53125.1
AD-52798.1
AD-53126.1
AD-52799.1
AD-53127.1
AD-52800.1
AD-53128.1
AD-52801.1
AD-53129.1
AD-52802.1
AD-53130.1
AD-52803.1
AD-53131.1
AD-52804.1
AD-53132.1
AD-52805.1
AD-53133.1
AD-52806.1
AD-53134.1
AD-52807.1
AD-53135.1
AD-52808.1
AD-53136.1
AD-52809.1
AD-53137.1
AD-52810.1
AD-53138.1
AD-52811.1
AD-53139.1
AD-52812.1
AD-53140.1
AD-52813.1
AD-53141.1
AD-52814.1
AD-53142.1
AD-52815.1
AD-53143.1
AD-52816.1
AD-53144.1
AD-52817.1
AD-53145.1
AD-52818.1
AD-53146.1
AD-52819.1
AD-53147.1
AD-52820.1
AD-53148.1
AD-52821.1
AD-53149.1
AD-52822.1
AD-53150.1
In Vivo Tests
Example 3
Test Articles
In vivo experiments were conducted using dsRNA sequences of the invention. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). The dsRNA sequence used as a negative control was luciferase-conjugated AD-48399B1 (“Luc”, sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). Also used as a negative control was GalNal-conjugated AD-1955 containing alternating 2′-methyl and 2′ fluoro modifications.
Experimental Procedure
The dsRNA sequences were tested in C57BL/6 (WT) and ob/ob mice. WT mice received five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5 or 20 mg/kg; and ob/ob mice received five daily doses of NPLs formulated with Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles were administered by subcutaneous injection according to the procedure shown in FIG. 1. Specifically, five daily doses of the test articles were administered on five consecutive days (day 0, 1, 2, 3 and 4), and blood samples were collected 5, 3 or 1 day prior to administration, as well as on days 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45 and 50 post-administration. The collected blood samples were used to measure the expression of ANGPTL3 protein using an ELISA assay. Levels of serum triglycerides (TGs), low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc) and total cholesterol (TC) were also measured using an Olympus Analyzer.
Results
Shown in FIG. 2, Panel A, are levels of murine ANGPTL3 (mANGPTL3, protein measured in WT mice after administration of control or ANG at 5 or 20 mg/kg. Also shown in FIG. 2, Panel B are levels of mANGPTL3 protein measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that, for both WT and ob/ob mice, administration of ANG results in decreased levels of mANGPTL3 protein, as compared to controls.
Shown in FIG. 3, Panel A, are levels of LDL-c measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 3, Panel B are levels of LDL-c measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of LDL-c, particularly in ob/ob mice, as compared to controls.
Shown in FIG. 4, Panel A, are levels of triglycerides measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 4, Panel B are levels of triglycerides measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of tryglycerides, particularly, in ob/ob mice, as compared to controls.
Shown in FIG. 5, Panel A and B are levels of total cholesterol (TC) measured in WT and ob/ob mice, respectively, after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes a moderate decrease in TC levels in ob/ob mice, but not in WT mice. Similarly, administration of ANG causes a moderate decrease in HDL-c levels in ob/ob mice, but not in WT mice, as is shown in the graphs in FIG. 6.
Example 4
Test Article
The effect of a single injection of dsRNA sequence of the invention on the level of ANGPTL3 protein was tested. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). PBS was used as a negative control.
Experimental Procedure
The dsRNA sequences were tested in Human PCS Transgenic mouse characterized by liver-specific expression of full-length human PCSK9 gene. Human PCS transgenic mice were dosed with the AD-52981 or PBS using a single subcutaneous injection. The mice were divided into four groups, each group consisting of two males and two females. Each group received an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose of AD-52981. Blood samples were collected at day 1 and day 0 prior to dosing, and at 72 hours post dosing. ANGPTL3 protein levels were measured by ELISA and compared to levels at dayl and day 0 prior to dosing.
Results
Shown in FIG. 7, are levels of murine ANGPTL3 protein (mANGPTL3) measured in Human PCS transgenic mice. The data shown is expressed relative to PBS control and represents an average for 2 males and 2 females in each group. Error bars represent standard deviation. The data indicates that administration of a single injection of AD-52981 reduces the levels of ANGPTL3 protein in the mice in a dose-dependent manner, with the dose of 60 mg/kg decreasing the levels of ANGPTL3 protein more than five-fold (see FIG. 7).
SEQUENCES
>gi|41327750|ref|NM_014495.2| Homo sapiens angiopoietin-like 3
(ANGPTL3), mRNA
SEQ ID NO: 1
TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCT
TCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTC
CAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGA
CATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATT
TGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAA
CTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAA
AGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAAT
TCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGATA
ATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATAGTCAAATA
AAAGAAATAGAAAATCAGCTCAGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCC
AAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG
CTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCT
CAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGG
ATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGT
TGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGG
AAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCT
AGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATC
ACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGA
GAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATC
TTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAG
AAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGT
GGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTAAAGTCACT
GTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATTCT
TATAATACTATTTGTTTTAAATTTTGTGATGTGGGAATCAATTTTAGATGGTCACAATCTAGATTATAATC
AATAGGTGAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATCATAT
GAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTAAACTTGACTAAATAC
AGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTATTAATTTCAAAACTAAAAATCGTCAGCACAGA
GTATGTGTAAAAATCTGTAATACAAATTTTTAAACTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAA
ATGTTTGATATGATTTATTTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGT
CTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATT
>gi|297278846|ref|XM_001086114.2| PREDICTED: Macaca mulatta
angiopoietin-like 3 (ANGPTL3), mRNA
SEQ ID NO: 2
ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGTTGCTTGAAATTGAAAATC
AGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGACCA
AGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAA
TTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCAAATT
AATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGTGAAAT
CAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATA
TGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAA
TATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACATCCAGAAGTAACTTC
ACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACAATATA
AGCAATTAAACCAACAGCACAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTCAAGAA
CCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAATGAAAT
AAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATATAAGTG
GCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTAAAACC
TGTCTAAGGAGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTCGGGAG
GCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTACGTTTTAC
GAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAA
ACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTT
GGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTATTCAGGAGGCTGGT
GGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAACAAAATCTAAGCCA
GAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTT
GATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAAATTAAACATTA
AACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGCTTGAGAAATAGATT
TTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCATTTAC
ATTTCTCAATCAAAATTCCTACAACACTATTTGTTTTATATTTTGTGATGTGGGAATCAATTTTAGATGGT
CGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAAACTTAGAGACTTTA
ATTTTAAAAGTCATCATATGAGCTAATATCACAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTTAAAAC
TCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTAATATTAATTTCAAA
ACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACTGATGCCTCATTTTG
CTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAGCAGGATTAAATACT
GTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCACATTGACTTTAATAT
GAGGTATCACTATACCTTA
>gi|142388354|ref|NM_013913.3| Mus musculus angiopoietin-like 3
(Angpt13), mRNA
SEQ ID NO: 3
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTC
CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC
AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC
ATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTT
GATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC
TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA
GTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT
CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAA
CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAA
AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA
AGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC
CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCC
AAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC
TCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTT
GGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA
AAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTG
GCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCA
CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG
AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC
TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGA
AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT
AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA
TCAGATATCACCGAATCAACGTAAACAAAATTTATC
>gi|68163568|ref|NM_001025065.1| Rattus norvegicus angiopoietin-like 3
(Angpt13), mRNA
SEQ ID NO: 4
GACGTTCCAAATTGCTTGAAATTGAATAATTGAAACAAAAATGCACACAATTAAGCTGCTCCTTTTTGTTG
TTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGACCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAAAA
TCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCTTAA
AGATTTTGTCCATAAGACAAAGGGACAAATTAATGACATATTTCAGAAGCTCAACATATTTGATCAGTGTT
TTTATGACCTATCACTTCAAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACATCTAAA
CTACAAGTTAAAAACGAAGAGGTGAAGAATATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCTACTGGA
GGAGAAGATGGCGCTCCAACACAGAGTCAGGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAGAACCCGC
CTGGGGCTCGGGAGCACCCAGAGGTAACGTCACTTAAAAGTTTTGTAGAACAGCAAGATAACAGCATAAGA
GAACTCCTCCAGAGTGTGGAAGAACAATATAAACAACTAAGTCAACAGCACATTCAGATAAAAGAAATAGA
AAATCAGCTCAGAAAGACTGGCATTCAAGAACCCACTGAAAATTCTCTTTATTCTAAACCAAGAGCACCAA
GAACTACTCCCCCTCTTCATCTGAAGGAAGCAAAAAATATAGAACAAGATGATCTGCCTGCTGACTGCTCT
GCCATTTATAACAGAGGTGAACATACAAGTGGCGTGTATACTATTAGACCAAGCAGCTCTCAAGTGTTTAA
TGTCTACTGTGACACCCAATCAGGCACTCCACGGACATTAATTCAACACCGGAAAGATGGCTCTCAAAACT
TCAACCAAACGTGGGAAAACTACGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGATTTCCTTGCATCAC
TCACTTATCTGTTGATTTAATAGTATTAGTTGGGTGTGTTGACACAGGCCTGAGACCATAGCGCTTTTGGG
CAAGGGGGGAGGAGGAGCAGCAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCACACATTGATACTCCTT
CTCGACATTAAGAATTATAAATTAAGCAGCAATTATAAAATGGGCTGTGGAAATGTAACAATAAGCAAAAG
CAGACCCCAGTCTTCATAAAACTGATTGGTAAATATTATCCATGATAGCAACTGCAATGATCTCATTGTAC
TTATCACTACTGCATGCCTGCAGTATGCTTGTTGAAACTTAATTCTATAGTTCATGGTTATCATAAGTCTT
ATTAAGGAACATAGTATACGCCATTGGCTCTAGTGAGGGGCCATGCTACAAATGAGCTGCAAAGATAGCAG
TATAGAGCTCTTTCAGTGATATCCTAAGCACAACGTAACACAGGTGAAATGGGCTGGAGGCACAGTTGTGG
TGGAACACGCGGCCAGCAGGACACTGGGACTGATCCCCAGCAGCACAAAGAAAGTGATAGGAACACAGAGC
GAGAGTTAGAAGGGACAGGGTCACCGTCAGAGATACGGTGTCTAACTCCTGCAACCCTACCTGTAATTATT
CCATATTATAAACATATACTATATAACTGTGGGTCTCTGCATGTTCTAGAATATGAATTCTATTTGATTGT
AAAACAAAACTATAAAAATAAGTAAAAAAATAAAAAATAAACAGATACTTAAAATCAAAAAAAAAAAAAAA
AAAAAAAAAA
Reverse Complement of SEQ ID NO: 1
SEQ ID NO: 5
AATAAGGTATAGTGATACCTCATGTTAAAGTCAATGTGACTTAGTAGTCATCTCCATTTGTTTAAAGACAG
CGAACTTATTTTAATACAGTATTTAATTCTGCTTCATTAGGTTTCATAAATAAATCATATCAAACATTTAC
TCCAAATTATTTTGTAGCAAAATGAAGCATCAGTTTAAAAATTTGTATTACAGATTTTTACACATACTCTG
TGCTGACGATTTTTAGTTTTGAAATTAATACTACAACATTTAAGAACTGTACAATTACCAGTCCTCTGTAT
TTAGTCAAGTTTAGAGTTTTAACAAGAGTACTAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCTCATAT
GATGCCTTTTAAAATAAAAGTCTCTAAATTTTTTATTTAGAAAAGTTATTTAATAAGTTCACCTATTGATT
ATAATCTAGATTGTGACCATCTAAAATTGATTCCCACATCACAAAATTTAAAACAAATAGTATTATAAGAA
TTTTGATTGAGAAATGTAAACGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATAGACAGTG
ACTTTAAGATAAAAAAAATCTATTTCTCAAGCTTTCTCTTAAGGATTTAATACCAGATTATTAGACCACAT
TAACTTGGAATGAGGTTAATGTTTAAATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTCTGA
ATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAAGATA
ATCCTCTTCTCCTCTCTGGCTTAGATTTTGCTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCTCCA
CACTCATCATGCCACCACCAGCCTCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTTTTGCTTTGTGATC
CCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCGCAACTAGAT
GTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTCCAG
TCTTCCAACTCAATTCGTAAAACATAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAACCA
AAATTCTCCATCAAGCCTCCCAAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATCCAT
CTATTCGATGTTGAATTAATGTCCATGGACTACCTGATATAACATCACAGTAGACATGAAAAACTTGAGAG
TTGCTGGGTCTGATGGCATACATGCCACTTGTATGTTCACCTCTGTTATAAATGGTGGTACATTCAGCAGG
AATGCCATCATGTTTTACATTTCTTATTTCATTCAACTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCT
TGGAAGATAGAGAAATTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGCTGATTTTCTATTTCTTTTATT
TGACTATGCTGTTGGTTTAATTGTTTATATTGGTCTTCCACGGTCTGGAGAAGGTCTTTGATGCTATTATC
TTGTTTTTCTACAAAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGGAGTTTCAGGTTGATTTTGAATTA
AGTTAGTTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCA
AGTTTTGAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCT
TCTCAGTTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGATAGATCATAAAAAGACTGATCAAATA
TGTTGAGTTTTTGAAATATGTCATTAATTTGGCCCTTCGTCTTATGGACAAAGTCTTTAAGACCATGTCCC
AACTGAAGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGA
TAGAGAATCAAATGATGAATTGTCTTGATCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGA
GCTTAATTGTGAACATTTTTATCTTGATTTTCAATTTCAAGCAACGTGGAACTGTTTTCTTCTGGAA
Reverse Complement of SEQ ID NO: 2
SEQ ID NO: 6
TAAGGTATAGTGATACCTCATATTAAAGTCAATGTGACTTAGTAATCATCATCTCCATTTGTTTAAAAGAC
AGCGAACCTATTTTAATACAGTATTTAATCCTGCTTCATTAGGTTTCATAAATAAATCCTATCAAAAATTT
ACTCCAGATTATTTTGTAGCAAAATGAGGCATCAGTTTAAAAATTTATATTACAGATTTTTACACATACTC
TGTGCTGACAATTTTTAGTTTTGAAATTAATATTACAACAAATTTAAGAACTGTATAATTATCAGTCCTCT
TTATTTAGTCAAGTTTAGAGTTTTAACAAGAAAACTAGTTTTTTAAACTGGGAAAATTGTGATATTAGCTC
ATATGATGACTTTTAAAATTAAAGTCTCTAAGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCACCTGTT
GATTATAATTTAGATTGCGACCATCTAAAATTGATTCCCACATCACAAAATATAAAACAAATAGTGTTGTA
GGAATTTTGATTGAGAAATGTAAATGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATTGAC
AGTGACTTTAAGATAAAAAAATCTATTTCTCAAGCCTTCTCTTAAGGATTTAATACCAGATTATTAAACCA
CATTAACTTGGAATGAGTTTAATGTTTAATTTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTC
TGAATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAGG
ATAATCCTCTTCTCCGCTCTGGCTTAGATTTTGTTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCT
CCACACTCATCATGCCACCACCAGCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTCCTTTTGCTTTGTG
ATCCCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCTTAACTA
CATGTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTC
CAGTCTTCCAACTCAATTCGTAAAACGTAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAA
CCAGAATTCTCCATCAAGCCTCCCGAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATC
CATCTATTCTCCTTAGACAGGTTTTACCTGATACAACATCACAGTAGACATGAAAAACTTGAGAGTTGCTG
GGTCTGATGGCATACATGCCACTTATATGTTCACCTCTATTGTAAATGGTGGTACAATCAGCAGGAATGCC
ATCATGTTTTACATTTCTTATTTCATTCAGCTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCTTGGAAG
ATAGAGAAATTTCTGTGGGTTCTTGAATATTAGTCATTCTGAGCTGATTTTCTATTTCTTTTATTTGACTG
TGCTGTTGGTTTAATTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGTCTTTGATGCTATTATCTTGTTT
TTCTACAAAACTTTTAAGTGAAGTTACTTCTGGATGTTCTGGAGTTTCAGGTTGATTTTGAATTAAGTTAG
TTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCAAGTTTT
GAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCTTCTCAG
TTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGTGATAGATCATAAAAAGACTGATCAAATATGTTGA
GTTTTTGAAATATGTCATTAATTTGGCCCTTAGTCTTATGGACAAAGTCTTTAAGACCATGTCCCAACTGA
AGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGATACAGA
ATCAAATGATGAATTGTCTTGGTCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGAGCTTAA
TTGTGAACATTTTTATCCTGATTTTCAATTTCAAGCAACGTGGAACTGTGTTCTTCTGGAAGCAGACCTAG
ACTTCTTAACTCTATATAT
Reverse Complement of SEQ ID NO: 3
SEQ ID NO: 7
CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTC
CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC
AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC
ATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTT
GATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC
TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA
GTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT
CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAA
CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAA
AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA
AGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC
CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCC
AAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC
TCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTT
GGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA
AAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTG
GCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCA
CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG
AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC
TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGA
AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT
AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA
TCAGATATCACCGAATCAACGTAAACAAAATTTATC
Reverse Complement of SEQ ID NO: 4
SEQ ID NO: 8
TTTTTTTTTTTTTTTTTTTTTTTTTGATTTTAAGTATCTGTTTATTTTTTATTTTTTTACTTATTTTTATA
GTTTTGTTTTACAATCAAATAGAATTCATATTCTAGAACATGCAGAGACCCACAGTTATATAGTATATGTT
TATAATATGGAATAATTACAGGTAGGGTTGCAGGAGTTAGACACCGTATCTCTGACGGTGACCCTGTCCCT
TCTAACTCTCGCTCTGTGTTCCTATCACTTTCTTTGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCTGGCC
GCGTGTTCCACCACAACTGTGCCTCCAGCCCATTTCACCTGTGTTACGTTGTGCTTAGGATATCACTGAAA
GAGCTCTATACTGCTATCTTTGCAGCTCATTTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTATACTAT
GTTCCTTAATAAGACTTATGATAACCATGAACTATAGAATTAAGTTTCAACAAGCATACTGCAGGCATGCA
GTAGTGATAAGTACAATGAGATCATTGCAGTTGCTATCATGGATAATATTTACCAATCAGTTTTATGAAGA
CTGGGGTCTGCTTTTGCTTATTGTTACATTTCCACAGCCCATTTTATAATTGCTGCTTAATTTATAATTCT
TAATGTCGAGAAGGAGTATCAATGTGTGGCCCAGACTGGTCTTGAACTTTCAATTCACCTGCTGCTCCTCC
TCCCCCCTTGCCCAAAAGCGCTATGGTCTCAGGCCTGTGTCAACACACCCAACTAATACTATTAAATCAAC
AGATAAGTGAGTGATGCAAGGAAATCACTTTACCATCAAGCCTCCCAAAACCCTTTTCGTAGTTTTCCCAC
GTTTGGTTGAAGTTTTGAGAGCCATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGCCTGATTGGGTGTC
ACAGTAGACATTAAACACTTGAGAGCTGCTTGGTCTAATAGTATACACGCCACTTGTATGTTCACCTCTGT
TATAAATGGCAGAGCAGTCAGCAGGCAGATCATCTTGTTCTATATTTTTTGCTTCCTTCAGATGAAGAGGG
GGAGTAGTTCTTGGTGCTCTTGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTTGAATGCCAGTCTTTCT
GAGCTGATTTTCTATTTCTTTTATCTGAATGTGCTGTTGACTTAGTTGTTTATATTGTTCTTCCACACTCT
GGAGGAGTTCTCTTATGCTGTTATCTTGCTGTTCTACAAAACTTTTAAGTGACGTTACCTCTGGGTGCTCC
CGAGCCCCAGGCGGGTTCTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCCTGACTCTGTGTTGGAGCGC
CATCTTCTCCTCCAGTAGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACATATTCTTCACCTCTTCGTTTT
TAACTTGTAGTTTAGATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTGATTTCATTGGTTTGAAGTGAT
AGGTCATAAAAACACTGATCAAATATGTTGAGCTTCTGAAATATGTCATTAATTTGTCCCTTTGTCTTATG
GACAAAATCTTTAAGACCATGACCCAGCTGCAGGAGGCCATTGGCTAAAATTTTGACATCATCCAACATAG
CAAATCTTGATTTTGGCTCTGACGGTACAGAATCAAATGGCGAAAGGTCTGGATCAACTCTGGACGAAATT
ACTAGAGGAACAACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTGTTTCAATTATTCAATTTCAAGCAAT
TTGGAACGTC
Macaca fascicularis angiopoietin-like 3 (Angpt13), mRNA
SEQ ID NO: 9
GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGCTGCTTGAAATTGA
AAATCAGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATT
GACCAAGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGT
AAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCC
AAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGT
GAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAA
GAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAG
TGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGCAACTCCAGAACATCCAGAAGTA
ACTTCACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACA
ATATAAGCAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTC
AAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAAT
GAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATAT
AAGTGGCACGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTA
GTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAA
TATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATC
TAATTACGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACT
TGGGAAATCACGAAACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCG
GAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTA
TTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAA
CAAAATCTAAGCCAGAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAA
TCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAA
TAAATTAAACATTAAACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGC
TTGAGAAATAGATTTTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAA
AGAATACCATTTACATTTCTCAATCAAAATTCTTACAACACTATTTGTTTTATATTTTGTGATGTGGGAAT
CAATTTTAGATGGTCGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAA
ACTTAGAGACTTTAATTTTAAAAGTCATCATATGAGCTAATGTCACAATTTTCCCAGTTTAAAAAACTAGT
TTTCTTGTTAAAACTCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTA
ATATTAATTTCAAAACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACT
GATGCCTCATTTTGCTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAG
CAGGATTAAATACTGTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCAC
ATTGACTTTAATATGAGGTATCACTATACCTTAACATATTTGTTAAAACGTATACTGTATACATTTTGTGT
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17531888
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alnylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 20th, 2022 03:05PM
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Apr 20th, 2022 03:05PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Mar 25th, 2008 12:00AM
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Mar 7th, 2003 12:00AM
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https://www.uspto.gov?id=US07348314-20080325
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Compositions and methods for inhibiting viral replication
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The present invention relates to a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is less that 30 nucleotides in length and which is substantially identical to at least a part of a 3′-untranslated region (3′-UTR) of a (+) strand RNA virus, such as HCV, as well as pharmaceutical compositions comprising the dsRNA, together with a pharmaceutically acceptable carrier. The pharmaceutical compositions are useful for treating infections and diseases caused by the replication or activity of the (+) strand RNA virus, as well as methods for inhibiting viral replication.
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7348314
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1. A method for inhibiting the replication of hepatitis C virus in a hepatitis C virus infected cell in vitro, which comprises introducing a double-stranded ribonucleic acid (dsRNA) into the cell, wherein the dsRNA comprises a sense RNA strand consisting of SEQ ID No. 4 and an antisense strand consisting of SEQ ID NO:5.
2. The method of claim 1, wherein the cell is a mammalian cell.
3. The method of claim 2, wherein the mammalian cell is a human cell.
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3
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RELATED APPLICATIONS
This application is a continuation-in-part of International Application No. PCT/EP02/11432, which designated the United States and was filed on Oct. 11, 2002, which claims the benefit of German Patent No. 101 50 187.0, filed on Oct. 12, 2001, German Patent No. 101 55 280.7, filed on Oct. 26, 2001, German Patent No. 101 58 411.3, filed on Nov. 29, 2001, German Patent No. 101 60 151.4, filed on Dec. 7, 2001, German Patent No. 101 63 098.0, filed on Dec. 20, 2001, EP Patent No. PCT/EP02/00151, filed on Jan. 9, 2002, and EP Patent No. PCT/EP02/00152, filed on Jan. 9, 2002. The entire teachings of the above application(s) are incorporated herein by reference.
FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use for inhibiting the replication of (+) strand RNA viruses, such as Hepatitis C virus, as well as treating viral-associated diseases.
BACKGROUND OF THE INVENTION
Positive or plus-strand RNA viruses share many similarities in genomic organization and structure, most notably a single-stranded coding RNA of positive polarity. Representative (+) strand RNA viruses include the picornaviruses, flaviviruses, togaviruses, coronaviruses, and caliciviruses. One clinically significant representative of the flavivirus family is the hepatitis C virus (HCV), the causative agent for hepatitis C. Hepatitis C is an often chronic inflammatory disease of the liver which typically results in fibrosis and liver cancer (Choo, et al., Science (1989) 244:359). Infection by HCV typically results from contact with contaminated blood or blood products.
During HCV replication, a replicative (minus) RNA strand is produced which serves as a template for generation of several coding (+) RNA strands. The HCV genome, which contains approximately 9600 nucleotides, is translated into a polyprotein consisting of approximately 3000 amino acids (Leinbach, et al., Virology (1994) 204:163-169; Kato, et al., FEBS Letters (1991) 280:325-328). This polyprotein subsequently undergoes post-translational cleavage, producing several proteins. Due to high genetic variability and mutation rates, the HCV comprises several distinct HCV genotypes that share approximately 70% sequence identity (Simmonds, et al., J. Gen. Virol., (1994) 75:1053-1061). Despite this hypervariability, there are three regions of the HCV genome that are highly conserved, including the 5′- and 3′-non-coding regions, known as the 5′-untranslated region (5′-UTR) and 3′-untranslated region (3′-UTR), respectively. These regions are thought to be vital for HCV RNA replication as well as translation of the HCV polyprotein. In general, treatment of HCV is complicated by its high mutation rate, as well as the mode of transmission and possibility of simultaneous infection with multiple HCV genotypes.
Hepatitis C has several clinical phases. The first phase (i.e., acute phase) begins with infection by HCV. During this early phase, it is possible to detect HCV-RNA in the serum of patients using polymerase chain reaction (PCR). However, because only about 25% of patients exhibit jaundice during this phase, most cases (75%) go undetected in the early stages. The inflammatory process, characterized by an increase in serum liver enzyme concentrations, begins approximately four weeks post infection. Although acute HCV infection is not malignant, the majority of patients (approximately 80%) develop chronic liver disease, characterized by a permanent elevation in the serum alanine aminotransferase level. Cirrhosis of the liver develops in more than 20% of patients with chronic HCV disease, which frequently leads to malignant hepatoma. Life expectancy following diagnosis of the malignant hepatoma is generally 12 months.
Current therapies to treat HCV infections have met with limited success, with only a minority of patients experiencing long-term improvement. The most prevalent treatment today involves specific cytokines known as interferons, particularly interferon-α (IFN-alpha) which reduces serum alanine aminotransferase levels in approximately 50% of patients. Unfortunately, serum levels of alanine aminotransferase usually return to elevated levels following termination of treatment, producing a number of adverse side effects (Dusheiko, et al., J Viral Hepatitis (1994) 1:3). Despite these problems, IFN-alpha is commonly used to reduce the risk of cirrhosis of the liver and malignant hepatoma. There is no currently available vaccine for HCV.
Although IFN-alpha remains the conventional approach, virologists have evaluated a number of potential alternative therapies, including the use of specific ribozymes to inhibit translation of viral protein. Welch et al. disclose a two vector-expressed hairpin ribozyme directed against HCV (Welch, et al., Gene Therapy (1996), 3(11):994). Lieber et al. report the removal of HCV-RNA in infected human hepatocytes through adenovirus-mediated expression of specific hammerhead ribozymes (Lieber, et al., Virology (1996), 70 (12):8782). WO 99/55847 report the degradation of 5′- and 3′-UTL regions of HCV-RNA, as well as the 5′-coding region for the nucleoprotein, using ribozymes. U.S. Pat. No. 5,610,054 discloses enzymatic nucleic acid molecules that can inhibit replication of HCV. Despite these efforts, the therapeutic value of ribozymes for treating HCV infections remains questionable, particularly in view of their low enzymatic activity.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature (2000), 404:293-296). When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi. WO 99/32619 (Fires et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.); Drosophilia (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200); and mammals (WO 00/44895, Limmer).
Despite significant advances in the field, there remains a need for an agent that can inhibit the replication of a virus in a host cell using the cell's own RNAi machinery. More specifically, an agent that has high biological activity and can provide long-term, effective inhibition of viral replication at a low dose, would be highly desirable. Compositions comprising such agents would be useful for treating a variety of viral infections, including HCV.
SUMMARY OF THE INVENTION
The present invention discloses double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the replication of a (+) strand RNA virus, such as a Hepatitis C Virus (HCV). In particular, the invention relates to a dsRNA having an RNA strand (the complementary strand) comprising a region which is complementary to at least a portion of a 3′-untranslated region (3′-UTR) of a (+) strand RNA virus. The present invention also discloses compositions and methods for treating hepatitis C or HCV-associated diseases.
In one aspect, the invention relates to a dsRNA. The dsRNA comprises a sense RNA strand comprising a nucleotide sequence which is substantially identical to at least a part of a 3′-untranslated region (3′-UTR) of a (+) strand RNA virus, and the dsRNA is less than 30 nucleotides in length. The (+) strand RNA may be a hepatitis C virus. The dsRNA may further comprise a complementary RNA strand, wherein the complementary RNA strand comprises a complementary nucleotide sequence which is less than 30 nucleotides in length and is complementary to at least a portion of the 3′-UTR of the virus. In a preferred embodiment, the nucleotide sequence is within a highly conserved region of the 3′-UTR. The complementary nucleotide sequence is preferably less than 25 nucleotides in length, more preferably 21 to 24 nucleotides in length, and most preferably 23 nucleotides in length. The dsRNA may comprise one or two blunt ends. The complementary RNA strand and the sense RNA strand may comprise a 3′-terminus and a 5′-terminus, and at least one of the RNA strands may comprise a nucleotide overhang of 1 to 3 nucleotides in length, preferably two nucleotides in length. The dsRNA may further comprise two ends, wherein one end comprises the 3′-terminus of the complementary RNA strand and the 5′-terminus of the sense RNA strand, and the other end comprises the 5′-terminus of the complementary RNA strand and the 3′-terminus of the sense RNA strand. In one embodiment, one end of the dsRNA end has a nucleotide overhang, preferably on the 3′-terminus of the complementary RNA strand, and the second end is blunt. In another embodiment, the complementary RNA strand is 24 nucleotides in length and the sense RNA strand is 22 nucleotides in length, the 3′-end of the complementary RNA strand has a 2-nucleotide overhang, and the other end of the dsRNA is blunt. In a particular embodiment, the complementary RNA strand comprises the nucleotide sequence of SEQ ID NO:5 and the sense RNA strand comprises the nucleotide sequence of SEQ ID NO:4.
In another aspect, the invention relates to a pharmaceutical composition for inhibiting the replication of a (+) strand RNA virus in an organism, such as a mammal (e.g., human). The pharmaceutical composition comprises the dsRNA as described above, together with a pharmaceutically acceptable carrier. The dosage unit of dsRNA in the composition may be less than 5 milligram (mg) of dsRNA per kg body weight, preferably 0.01 to 2.5 milligrams (mg), more preferably 0.1 to 200 micrograms (μg), and most preferably 0.1 to 100 μg per kilogram body weight. In one embodiment, the pharmaceutically acceptable carrier is an aqueous solution, such as phosphate buffered saline. In another embodiment, the pharmaceutically acceptable carrier comprises a micellar structure, such a liposome, capsid, capsoid, polymeric nanocapsule, or polymeric microcapsule. The pharmaceutical composition may be formulated to be administered by inhalation, infusion, injection, or orally. In one embodiment, the pharmaceutical compositions is formulated to be administered by intravenous or intraperitoneal injection.
In yet another aspect, the invention relates to a method for inhibiting the replication of a (+) strand RNA virus comprising a 3′-untranslated region (3′-UTR) in a cell. The method comprises introducing a double-stranded ribonucleic acid (dsRNA), as described above, into the cell. The dsRNA comprises a nucleotide sequence which is substantially identical to at least a part of the 3′-UTR, and the dsRNA is less than 30 nucleotides in length, more preferably less than 25 nucleotides, more preferably 21 to 24 nucleotides, and most preferably 23 nucleotides in length.
In still another aspect, the invention relates to a method for treating a disease associated with infection of a (+) strand RNA virus in an organism. The method comprises administering a pharmaceutical composition to the organism, wherein the pharmaceutical composition comprises a double-stranded ribonucleic acid (dsRNA), as described above, together with a pharmaceutically acceptable carrier. The dsRNA comprises a nucleotide sequence which is substantially identical to at least a part of the 3′-UTR of the (+) strand RNA virus, and the dsRNA is less than 30 nucleotides in length.
The details of once or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the relevant sequence region (SEQ ID NO: 2) from the p2 plasmid and the N-terminal amino acid sequence (SEQ ID NO:17) of the corresponding reporter protein.
FIG. 2 shows the relevant sequence region (SEQ ID NO: 3) from the p3 plasmid and the N-terminal amino acid sequence (SEQ ID NO:18) of the corresponding reporter protein.
FIG. 3 shows the HCV1-2 dsRNA (SEQ ID NO 4, SEQ ID NO 5) in contrast to the HCV sequence of an mRNA (SEQ ID NO 15) formed by means of the p2 and p3 plasmids.
FIG. 4 shows the GAL1-2 dsRNA (SEQ ID NO 6, SEQ ID NO 7) in contrast to the mRNA sequence (SEQ ID NO 16) corresponding to β-gal gene (positive control).
FIG. 5 shows the HCV3-4 the dsRNA(SEQ ID NO 8, SEQ ID NO 9), that exhibits no relation to the expressed genes (negative control).
FIG. 6 shows the K22 dsRNA (SEQ ID NO 10, SEQ ID NO 11), that exhibits no relation to the expressed genes (negative control).
FIG. 7 shows the antisense oligonucleotides HCVPT01 (SEQ ID NO 12), HCVPTO2 (SEQ ID NO 13), and HCVPTO3 (SEQ ID NO 14), in comparison to the HCV sequence of mRNA (SEQ ID NO 15) formed by the p3 plasmid.
FIG. 8 shows the effect of various concentrations of HCV1-2, GAL1-2, and HCV3-4 dsRNAs on the activity of β-galactosidase expressed by means of the p2 plasmid.
FIG. 9 shows the effect of various concentrations of HCV1-2, GAL-2, and HCV3-4 dsRNAs on the activity of β-galactosidase expressed by means of the p3 plasmid.
FIG. 10 shows the effect of the antisense oligonucleotides HCVPTO1, HCVPTO2, and HCVPTO3 of dsRNAs HCV1-2, GAL1-2, and HCV3-4 on the activity of β-galactosidase expressed by means of the p3 plasmid.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the replication of a (+) strand RNA virus, such as a Hepatitis C Virus (HCV), using the dsRNA. The present invention also discloses compositions and methods for treating diseases in organisms caused by infection with HCV or HCV-associated diseases. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates. The dsRNA of the invention comprises an RNA strand (the complementary strand) having a region that is complementary to at least a portion of a 3′-untranslated region (3′UTR) of a (+) strand RNA virus. Using a cell-based assay, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi in mammalian cells, resulting in a significant reduction in the activity or level of RNA encoded by the HCV genome as compared to untreated control cells. The present invention encompasses these dsRNAs and compositions comprising dsRNA and their use for specifically inhibiting the activity or replication of a (+) strand RNA virus such as HCV. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in (+) RNA strand viral infections, including Hepatitis C. Thus, the methods and compositions of the present invention comprising these dsRNAs are useful for treating HCV and HCV-associated diseases.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the activity or replication of a (+) strand RNA virus, as well as compositions and methods for treating viral diseases. The pharmaceutical compositions of the present invention comprise a dsRNA having a complementary nucleotide sequence of less than 30 nucleotides in length, preferably less than 25 nucleotides in length, and most preferably 21 to 24 nucleotides in length, and which is substantially identical to at least a part of a 3′-UTR of a (+) strand RNA virus, together with a pharmaceutically acceptable carrier. The dsRNA is less than 30 nucleotides in length, preferably less than 25 nucleotides in length, and most preferably 21 to 24 nucleotides in length. The dsRNA may be blunt ended, or one end, preferably the 3′-end of the complementary (antisense) strand, may have a single-stranded nucleotide overhang of 1 to 3 nucleotides, preferably 2 nucleotides in length.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the dsRNA of the present invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit the activity or replication of (+) strand RNA viruses such as HCV, and methods of using the pharmaceutical compositions to treat Hepatitis C and HCV-associated diseases.
I. Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.
As used herein, the terms “3′-untranslated region” and “3′-UTR” refer to the conserved, non-coding region at the 3′-end of a viral genome. The 3′-UTR can be the entire non-coding region or a fragment thereof. As used herein, the term “highly conserved region” refers to a region of the viral genome that remains evolutionarily constant, i.e., a genomic region that has a very low mutation rate and thus shares significant sequence identity (>99%) between distinct viral genotypes.
The term “complementary RNA strand” (also referred to herein as the “antisense strand”) refers to the strand of a dsRNA which is complementary to a 3′-UTR of a (+) strand RNA virus. As used herein, the term “complementary nucleotide sequence” refers to the region on the complementary RNA strand that is complementary to the 3′-UTR. “dsRNA” refers to a ribonucleic acid molecule having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. Not all nucleotides of a dsRNA must exhibit Watson-Crick base pairs; the two RNA strands may be substantially complementary (i.e., having no more than one or two nucleotide mismatches). The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. The RNA strands may have the same or a different number of nucleotides. Similarly, the complementary nucleotide sequence is less than 30, preferably less than 25, and most preferably 21 to 24 nucleotides in length. The dsRNA is also preferably less than 30, more preferably less than 25, and most preferably 21 to 24 nucleotides in length. Thus, the length of the dsRNA preferably corresponds to the length of the complementary nucleotide sequence. “Introducing into” means uptake or absorption in the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through cellular processes, or by auxiliary agents or devices. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro delivery includes methods known in the art such as electroporation and lipofection.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure when a 3′-end of one RNA strand extends beyond the 5′-end of the other complementary strand, or vice versa. “Blunt” or “blunt end” means that the lengths of the two RNA strand are the same at that end of the dsRNA, and hence there is no nucleotide(s) protrusion (i.e., no nucleotide overhang).
As used herein and as known in the art, the term “identity” is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math.(1988) 48:1073. “Substantially identical,” as used herein, means there is a very high degree of homology (preferably 100% sequence identity) between the sense strand of the dsRNA and the corresponding part of the target 3′-UTR of the viral genome. However, dsRNA having greater than 90%, or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. Although 100% identity is preferred, the dsRNA may contain single or multiple base-pair random mismatches between the RNA and the target 3′-UTR.
As used herein, the term “treatment” refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier or diluent for administration of a therapeutic agent. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed. 1985), which is hereby incorporated by reference herein. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a dsRNA molecule has been introduced by means of recombinant DNA techniques.
II. Double-stranded Ribonucleic Acid (dsRNA)
In one embodiment, the invention relates to a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is substantially identical to at least a portion of a target 3′-UTR of a (+) strand RNA virus. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form the duplex structure. One strand of the dsRNA comprises the nucleotide sequence that is substantially identical to a portion of the target 3′-UTR (the “sense” strand), and the other strand (the “complementary” or “antisense” strand) comprises a sequence that is complementary to the 3′-UTR. Because of this complementarity, the complementary RNA strand is able to base-pair with the complementary region of the 3′-UTR, thus inducing a structural change within the target 3′-UTR. For example, the complementary region of the 3′-UTR may be cleaved (through RNA interference) and/or ligated to other nucleic acid molecules, thus resulting in degradation and/or insertion or deletion mutations. Binding between the complementary RNA strand and the target 3′-UTR can also induce a structural change in the secondary and/or tertiary structure of the 3′-UTR. Because this region is vital for viral replication, such structural changes can block or significantly inhibit replication. Moreover, due to the high sequence variability of the genome of (+) strand RNA viruses, particularly HCV, sdRNAs that target conserved regions of the 3′-UTR may have a significant impact over a wide range of viral genotypes. Thus, not only is the efficiency of inhibition of viral replication increased by targeting a highly conserved region of the 3′-UTR, but targeting such regions also enables the treatment of diverse patient populations.
The sequence that is complementary to the target 3′-UTR (i.e., the complementary nucleotide sequence) is less than 30 nucleotides, preferably less than 25 nucleotides, and most preferably 21 to 24 nucleotides in length. Similarly, the dsRNA may have less than 30 nucleotides, preferably less than 25 nucleotides, and most preferably 21 to 24 nucleotides in length. The dsRNA can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer, such as are commercially available from Biosearch, Applied Biosystems, Inc. In specific embodiments, the dsRNA can comprise the sequence set forth in SEQ ID NOS:12 or 13, or a complement thereof. In a particular embodiment, the antisense (complementary) RNA strand comprises the sequence set forth in SEQ ID NO:5, and the sense RNA strand comprises the sequence set forth in SEQ ID NO:4.
In one embodiment, at least one end of the dsRNA is blunt. dsRNA with at least one blunt end show improved stability as compared to dsRNA having two nucleotide overhangs. dsRNA with at least one blunt end shows greater in vivo stability (i.e., is more resistant to degradation in the blood, plasma, and cells). However, dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without effecting its overall stability. The stability, particularly plasma stability, can thus be adjusted in accordance with needs of the particular application. dsRNA having only one overhang has proven particularly effective in vivo (as well as in a variety of cells, and cell culture mediums), and are more stable than dsRNA having two blunt ends. The single-stranded nucleotide overhang may be 1 to 3, preferably two, nucleotides in length. Preferably, the single-stranded overhang is located at the 3′-end of the complementary (antisense) RNA strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Preferably, the complementary strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt.
III. Pharmaceutical Compositions Comprising dsRNA
In one embodiment, the invention relates to a pharmaceutical composition comprising a dsRNA, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the dsRNA is useful for treating an infection or disease associated with the activity or replication of a (+) strand RNA virus.
The pharmaceutical compositions of the present invention are administered in dosages sufficient to inhibit the activity or replication of a (+) strand RNA virus, such as HCV. The present inventors have found that compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight per day is sufficient to inhibit or completely suppress the activity or replication of the target virus.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 2.5 milligrams per kilogram body weight of the recipient per day, more preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, and most preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the infection or disease, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases. For example, mouse repositories can be found at The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Network and at the European Mouse Mutant Archive. Such models may be used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.
For oral administration, the dsRNAs useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of dsRNA in the cells that harbor the virus. Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Surprisingly, the present inventors have discovered that compositions containing only naked dsRNA and a physiologically acceptable solvent are taken up by cells, where the dsRNA effectively inhibits replication of the virus. Although microinjection, lipofection, viruses, viroids, capsids, capsoids, or other auxiliary agents are required to introduce dsRNA into cell cultures, surprisingly these methods and agents are not necessary for uptake of dsRNA in vivo. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the dsRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075, which are incorporated by reference herein.
Toxicity and therapeutic efficacy of dsRNAs can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs useful according to the invention can be administered in combination with other known agents effective in treating viral infections and diseases. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
For oral administration, the dsRNAs useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
IV. Methods for Treating Viral Infections and Diseases
In one embodiment, the invention relates to a method for treating a subject having an infection or a disease associated with the replication or activity of a (+) strand RNA virus having a 3′-UTR, such as HCV. In this embodiment, the dsRNA can act as novel therapeutic agents for inhibiting replication of the virus. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that viral replication is inhibited. Because of their high specificity, the dsRNAs of the present invention specifically target (+) strand RNA viruses having a 3′-UTR, as described above, and at surprisingly low dosages.
Examples of (+) strand RNA viruses which can be targeted for inhibition include, without limitation, picornaviruses, caliciviruses, nodaviruses, coronaviruses, arteriviruses, flaviviruses, and togaviruses. Examples of picornaviruses include enterovirus (poliovirus 1), rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus), cardiovirus (encephalomyocarditis virus), aphthovirus (foot-and-mouth disease virus O), and parechovirus (human echovirus 22). Examples of caliciviruses include vesiculovirus (swine vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease virus), “Norwalk-like viruses” (Norwalk virus), “Sapporo-like viruses” (Sapporo virus), and “hepatitis E-like viruses” (hepatitis E virus). Betanodavirus (striped jack nervous necrosis virus) is the representative nodavirus. Coronaviruses include coronavirus (avian infections bronchitis virus) and torovirus (Beme virus). Arterivirus (equine arteritis virus) is the representative arteriviridus. Togavirises include alphavirus (Sindbis virus) and rubivirus (Rubella virus). Finally, the flaviviruses include flavivirus (Yellow fever virus), pestivirus (bovine diarrhea virus), and hepacivirus (hepatitis C virus). In a preferred embodiment, the virus is hepacivirus, the hepatitis C virus. Although the foregoing list exemplifies vertebrate viruses, the present invention encompasses the compositions and methods for treating infections and diseases caused by any (+) strand RNA virus having a 3′-UTR, regardless of the host. For example, the invention encompasses the treatment of plant diseases caused by sequiviruses, comoviruses, potyviruses, sobemovirus, luteoviruses, tombusviruses, tobavirus, tobravirus, bromoviruses, and closteroviruses.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.
V. Methods for Inhibiting Expression of a Target Gene
In yet another aspect, the invention relates to a method for inhibiting the replication or activity of a (+) strand RNA virus, such as HCV. The method comprises administering a composition of the invention to the host organism such that replication of the target virus is inhibited. The organism may be an animal or a plant. Because of their high specificity, the dsRNAs of the present invention specifically target (+) strand RNA viruses having a 3′-UTR, and at surprisingly low dosages. Compositions and methods for inhibiting the replication of a target virus using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of a 3′-UTR of a (+) strand RNA virus. When the organism to be treated is a mammal, such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous or intraparenteral infusion or injection.
The methods for inhibiting viral replication can be applied to any (+) strand RNA virus, such as those described above
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Inhibition of the 3′-UTR of HCV
To study RNA interference and the action of antisense oligonucleotides in a nonpathogenic assay, Sequence No. 1 in the sequence protocol was cloned in front of a gene that codes for E. coli β-galactosidase. Sequence No. 1 corresponds to a sequence from a highly conserved region of the 3′-UTR of the HCV genome that is 24 nucleotides in length. After transfection of the 3′-UTR plasmid in human HuH-7 liver cells, the sequence was transcribed as a part of an mRNA that codes for β-galactosidase. The mRNA sequence that corresponds to the 3′UTR is therefore identical to the HCV genome sequence and was subsequently used as the target sequence.
Generation of p2 and p3 Reporter Plasmids
The E. coli β-galactosidase (β-gal) gene was isolated from the commercially available expression vector pβ-Gal control (BD Biosciences Clontech, Tullastr. 4, 69126 Heidelberg, Germany, Gene Accession No. U13186, Nucleotide 280-3429).
The HCV sequence is part of a fusion gene in the p2 plasmid. The HCV sequence is part of the open reading frame of the sequence that codes for β-galactosidase, so that the HCV sequence is also expressed as part of a fusion protein. FIG. 1 shows the relevant sequence segments of the p2 plasmid (Sequence No. 2 of the sequence protocol). The HCV sequence is shown in italics. The beginning of the β-Gal gene (including 6 nucleotides of the Kozak sequence in front of the ATG codon) is underlined. The N-terminal amino acid sequence of the HCV β-galactosidase fusion protein is listed under the DNA sequence.
The HCV sequence is also part of a fusion gene in the p3 plasmid. However, the HCV sequence is located outside of the open reading frame of the sequence that codes for β-galactosidase, so that the HCV sequence is not expressed as part of a fusion protein. FIG. 2 shows the relevant sequence segment of the p3 plasmid (Sequence No. 3 in the sequence protocol). The HCV sequence is shown in italics. The beginning of the β-Gal gene (including 6 nucleotides of the Kozak sequence in front of the ATG codon) is underlined. The N-terminal amino acid sequence of the expressed β-galactosidase is listed under the DNA sequence.
The fusion genes that were generated in this way were cloned into the commercially available pcDNA3.1 (+) expression plasmid (Invitrogen, Life Technologies, Karlsruhe Technology Part, Emmy Noether Str. 10, 76131 Karlsruhe, Germany; Catalogue No. V790-20). This plasmid contains a neomycin resistance gene and thus confers on the HuH-7 cells that are transfected with it resistance to the G418. HuH-7 cells selected in the presence of G418 therefore harbor a reporter plasmid that stably integrated into the cell's genome. The commercially available pGL3-ctrl plasmid (Promega GmbH, High Tech Park, Schildkrötstr. 15, 68199 Mannheim, Germany; Gene Accession No. U47296 was used as the control plasmid. It codes and expresses the “firefly luciferase” gene.
Synthesis and Preparation of dsRNAs
Oligoribonucleotides are synthesized with an RNA synthesizer (Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and purified by High Pressure Liquid Chromatography (HPLC) using NucleoPac PA-100 columns, 9×250 mm (Dionex Corp.; low salt buffer: 20 mM Tris, 10 mM NaClO4, pH 6.8, 10% acetonitrile; the high-salt buffer was: 20 mM Tris, 400 mM NaClO4, pH 6.8, 10% acetonitrile. flow rate: 3 ml/min). Formation of double stranded dsRNAs is then achieved by heating a stoichiometric mixture of the individual complementary strands (10 M) in 10 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl, to 80-90° C., with subsequent slow cooling to room temperature over 6 hours.
In addition, dsRNA molecules with linkers may be produced by solid phase synthesis and addition of hexaethylene glycol as a non-nucleotide linker (D. Jeremy Williams, Kathleen B. Hall, Biochem. (1996) 35:14665-14670). A Hexaethylene glycol linker phosphoramidite (Chruachem Ltd, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, G20 OUA, Scotland, UK) is coupled to the support bound oligoribonucleotide employing the same synthetic cycle as for standard nucleoside phosphoramidites (Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany) but with prolonged coupling times. Incorporation of linker phosphoramidite is comparable to the incorporation of nucleoside phosphoramidites.
DsRNA Oligonucleotides
Three short double-stranded ribonucleic acids (dsRNA) were used for the RNA interference. These dsRNAs each consist of 2 short oligoribonucleotides that are complementary to each other over almost the entire sequence region. Two nucleotides have no base pairing at either of the 3′-ends of the oligoribonucleotides, and therefore form dsRNA overhangs.
The sequence of one of the oligoribonucleotides is identical to the mRNA target sequence. This oligoribonucleotide is therefore called the sense strand. The sequence of the other oligoribonucleotide is complementary to the mRNA target sequence. This oligoribonucleotide is therefore called the antisense strand.
The double-stranded oligoribonucleotide designated as HCV1-2 is shown in FIG. 3 and compared to the HCV sequence of the mRNA formed by means of the p2 and p3 plasmids. The nucleotides shown in capital letters correspond to the HCV sequence in the p2 and p3 plasmids. HCV1-2 consists of the HCV 1 sense strand and the HCV 2 antisense strand, whereby two nucleotides in each exhibit no base pairing at the 3′-ends of the strands. The sense strand (HCV 1) depicted in Sequence No. 4 in the sequence protocol exhibits almost the same nucleotide sequence as the HCV sequence of an mRNA formed by means of the p2 and p3 plasmids, respectively. Three nucleotides of the HCV sequence are missing at the 5′-end, and two nucleotides are present at the 3′-end that are not a component of the HCV sequence. The antisense strand (HCV 2) depicted in Sequence No. 5 in the sequence protocol is, except for the two nucleotides at the 3′-end, complementary to HCV 1, and therefore also to the HCV sequence of an mRNA formed by means of the p2 and p3 plasmids, respectively. The HCV sequence corresponds to a 3′-untranslated region of the HCV genome.
A dsRNA designated as GAL1-2 was used as the positive control. It is shown in FIG. 4 in contrast to an mRNA sequence (designated as mRNA in FIG. 4) that corresponds to the β-Gal gene of the p2 and p3 plasmids. GAL 1-2 consists of the Gal 1 sense strand and the Gal 2 antisense strand, whereas two nucleotides in each exhibit no base pairing at the 3′-ends of the strands. The sense strand (Gal 1) shown in Sequence No. 6 in the sequence protocol exhibits almost the same nucleotide sequence as the mRNA sequence that corresponds to the β-Gal gene. The antisense strand (Gal 2) shown in Sequence No. 7 in the sequence protocol is, except for the two nucleotides at the 3′-end, complementary to Gal 1, and therefore also to the mRNA sequence that corresponds to the β-Gal gene.
In one part of the experiment, a dsRNA designated as HCV3-4, which has no relationship to the genes expressed here, was used as the negative control (FIG. 5). HCV3-4 consists of the HCV 3 sense strand and the HCV 4 antisense strand, whereby two nucleotides in each exhibit no base pairing at the 3′-ends of the strands. The sense strand (HCV 3) shown in Sequence No. 8 of the sequence protocol exhibits almost no similarity to the mRNA formed by means of the p2 and p3 plasmids, and therefore has no relationship to the expressed genes. The antisense strand (HCV 4) shown in Sequence No. 9 in the sequence protocol is, except for the two nucleotides at the 3′-end, complementary to HCV 3 and therefore also has no relationship to the mRNA that is formed.
In another part of the experiment, a dsRNA designated as K22 was used as the negative control. It also exhibits no relationship to the gene expressed here (FIG. 6). The sequences of both oligoribonucleotides that form the dsRNA are shown in Sequence Nos. 10 and 11 in the sequence protocol.
Three 21-nucleotide-long DNA antisense oligoribonucleotides were used as phosphothioates in the experiments on antisense oligoribonucleotides. The oligoribonucleotides were obtained from Metabion GmbH, Lena-Christ Str. 44, 82152 Martinsried, Germany. They are here designated as HCVPTO1, HCVPTO2, and HCVPTO3. HCVPTO1 and HCVPTO2 are complementary to different regions of the HCV-mRNA sequence formed by means of the p3 plasmid. HCVPTO3 is the negative control without relationship to the target sequence. HCVPTO1, HCVPTO2, and HCVPTO3 are shown in FIG. 7 in contrast to the HCV-mRNA sequence. RNA interference assays were tested on the HuH-7 type liver cell line (Nakabayashi et al. 1982 which can be infected by HCV and is used routinely to culture these viruses. The cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) with 10% fetal calf serum (FCS).
a) Experiments relating to RNA interference
Transfection
Prior to transfection, 2×104 cells per well of a 96-well cell culture plate were seeded. 3 μg p2 plasmid and p3 plasmid, respectively, were mixed with 1 μg pGL3-ctrl plasmid. 0.25 μg of this plasmid mixture was placed in each well for transfection. Approximately 24 hours after seeding the cells, the p2/pGL3-ctrl and p3/pGL3-ctrl reporter plasmids were transfected together with dsRNA in HuH-7. The quantity of transfected DNA per well was constant.
The dsRNA was added to the plasmid mixtures in decreasing concentrations of 400 nmol/l to 12.5 nmol/l (in relation to 110 μl total transfection volume). The initial concentration of the HCV1-2, GAL1-2, and nonspecific HCV3-4 dsRNAs in each stock solution was 20 μmol/l. The dsRNAs were diluted by mixing them stepwise with the same volume of annealing buffer (AB, 100 mmol/l NaCl, 20 mmol/l sodium phosphate, pH 6.8) to arrive at the end concentration.
For an end concentration of 400 nmol/l, 2.2 μl stock solution was used for a transfection volume of 110 μl per well, and 6.6 μl stock solution was used for a transfection volume of 330 μl per well, respectively. The dilution steps were produced as shown in Table 1.
TABLE 1
Production of dsRNA dilution steps
Concentra-
Quantity
End con-
Solu-
tion of
of initial
Quantity
centra-
tion
initial solu-
solution
of added
tion *
No.
Initial solution
tion (μmol/l)
(μl)
AB (μl)
(nmol/l)
1
Stock solution
20
14.0
400
2
Solution 1
10
7.0
7.0
200
3
Solution 2
5
7.0
7.0
100
4
Solution 3
2.5
7.0
7.0
50
5
Solution 4
1.25
7.0
7.0
25
6
Solution 5
0.62
7.0
7.0
12.5
* End concentration, using 6.6 μl of each solution to a transfection volume of 330 μl
Plasmids and dsRNA were cotransfected. Gene Porter 2 (PeQLab, Carl Thiersch Str. 2B, 91052 Erlangen, Germany; Catalogue No. 13-T202007) was used as the transfection agent. Each cotransfection was repeated three times.
For 3 wells of the 96-well plates a mixture was made that consisted of 2.0 μl of a plasma mixture consisting of the p2 plasmid and the pGL3 control plasmid (0.3875 μg/μl; 3:1), 6.6 μl dsRNA (20, 10, 5, 2.5, 12.5, and 0.62 μmol/l, respectively), and 16.4 μl DNA diluent B (supplied together with Gene Porter 2, PeQLab). This mixture was mixed with a mixture consisting of 6.0 μl Gene Porter 2 and 19 μl serum-free medium. The total volume of the resultant mixture was 50 μl, of which 16.5 μl was added to each of 2×104 HuH-7 in 100 μl of medium. Then a mixture was made that consisted of 2.0 μl of a plasmid mixture consisting of the p3 plasmid and the pGL3 control plasmid (0.3875 μg/μl; 3:1), 6.6 μl dsRNA (20, 10, 5, 2.5, 12.5, and 0.62 μmol/l, respectively), and 16.4 μl DNA diluent B. This mixture was mixed with a mixture consisting of 6.0 μl Gene Porter 2 and 19 μl serum-free medium. The total volume of the resultant mixture was 50 μl, of which 16.5 μl was added to each of 2×104 HuH-7 in 100 μl of medium. The transfected cells were incubated at 37° C. and 5% CO2. 35 μl of fresh medium was added to each well, and the cells were incubated for another 24 hours. The cells were then trypsinied
Detection Methods Used
The effect of dsRNA on the expression of the reporter genes was determined by quantifying the β-galactosidase and luciferase activity by means of chemoluminescence. For this, lysates were made using the Tropix Lysebuffer (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif. 944404; Catalogue No. BD100LP) in accordance with manufacturer's instructions.
To quantify β-galactosidase activity, 2 μl lysate was used per analysis, as well as the substrate Galacto Star (Applied Biosystems, Tropix; Catalogue No. BM100S), in accordance with manufacturer instructions. To quantify luciferase activity, 5 μl lysate was used per analysis, as well as the substrate Luciferin (Applied Biosystems, Tropix; Catalogue No. BM100L) in accordance with manufacturer instructions. Luminescence was measured in each case using the Berthold Sirius luminometer (Berthold Detection Systems GmbH, Bleichstr. 56-58, 75173 Pforzheim, Germany).
Results
For each transfection assay, three 96-well plates were analyzed, such that in each case both β-galactosidase and luciferase were measured. The quotient of the relative light units (RLU) of β-galactosidase and the relative light units of luciferase were calculated. An average was determined for these three values. The average for p2/pGL3- and p3/pGL3 transfected cells without dsRNA, respectively, was arbitrarily defined as 1.0. The values that changed under the influence of dsRNA were recorded as a ratio to 1.0 (see FIGS. 8 and 9), i.e., a value of 0.6 corresponds to a 40% inhibition of β-galactosidase activity in comparison with untreated cells. In FIG. 8, cotransfection of sequence-specific dsRNA with the p2 plasmid resulted in a reduction in β-galactosidase activity. The HCV 1-2 and GAL 1-2 dsRNAs inhibit β-galactosidase with comparable effectiveness. At transfection volumes of 400 nmol/l and 200 mol/l of dsRNA, β-galactosidase activity decreases to 40% as compared to untreated cells. The inhibitory effect decreased with decreasing dsRNA concentration. The HCV3-4 control dsRNA leads to no decrease in β-galactosidase activity in lysate over the entire concentration range. A reduction in β-galactosidase expression is also detectable with cotransfection of the sequence-specific HCV1-2 dsRNA with the p3 plasmid (FIG. 9). HCV1-2 and GAL1-2 inhibit β-galactosidase activity with comparable effectiveness. At transfection volumes of 400 nmol/l and 200 mol/l of dsRNA, β-galactosidase activity decreases to approximately 20% as compared to untreated cells. The inhibitory effect decreased with decreasing dsRNA concentration. The HCV3-4 control dsRNA showed a weak inhibition of β-galactosidase activity to approximately 70% as compared to untreated cells. In the presence of the HCV1-2 dsRNA, both the p2 and p3 plasmids showed a marked decrease in β-galactosidase activity. Comparable effects were seen with the GAL1-2 dsRNA (positive control). The second control dsRNA, HCV3-4, led to no and markedly less inhibition of β-galactosidase activity, respectively. Expression and/or stability of RNA was markedly decreased by dsRNA in the experiments described. This was also true for HCV target sequences outside the open reading frame, which corresponds to the situation with the natural 3′-UTR region of HCV.
b) Experiments with antisense DNA oligonucleotides
To prepare for the experiments, p3 was stably transfected into HuH-7 cells using LipofectaminePLUS (GIBCO BRL Life Technologies, Karlsruhe Technology Park, Emmy Noether Str. 10, 76131 Karlsruhe, Germany). For this, 2×104 cells were seeded per well of a 96-well cell culture plate. After 24 hours, the medium was replaced with 50 μl serum-free medium (DMEM). The transfection mixture consisted of 0.2 μg p3, 16.7 μl DMEM, 2 μl PLUS reagent, and 1 μl Lipofectamine reagent. Cells were transfected in accordance with manufacturer's instructions. After three hours, the transfection medium was replaced with 150 μl complete medium (DMEM+10% fetal calf serum). After 48 hours, the cells were transferred to wells in a 12-well cell culture plate, and cultured with 400 μg/ml G418 (Amersham Biosciences, Munzinger Str. 9, 79111 Freiberg, Germany). Colonies were collected and transferred to new wells in a 12-well cell culture plate. From these, the cells that grew in the new wells after 14-21 days were culled manually and cultured with 400 μg/ml G418 until the selection was comkplete. After approximately three manual selections, β-galactosidase activity was determined as described below by means of enzyme measurements. Then the number of cells that expressed galactosidase was determined using X-Gal staining. For this, the medium was aspirated and the cells were stained in the wells of a 96-well cell culture plate overnight in 100 μl X-Gal solution (10 mmol/l sodium phosphate, pH 7.0; 1 mmol/l MgCL2; 150 mmol/l NaCl; 3.3 mmol/l K4Fe(CN)6*3H2O; 3.3 mmol/l K4Fe(CN)6; 0,2% X-Gal) (X-Gal from PeQLab, Erlangen, Germany; all other chemicals from SIGMA, Grünwalder Weg 30, 82024 Taufkirchen, Germany). The best clone was designated “HuH-7 blue” and used for the experiments.
Transfection with dsRNA and Antisense DNA Oligonucleotides
To prepare for a transfection, 2×104 cells of HuH-7 blue was seeded in 100 μl DMEM+10% FCS per well of a 96-well cell culture plate. After 24 hours, the dsRNA and the antisense DNA oligonucleotides were transfected. Fugene 6 (Roche Applied Sciences, Sandhofer Str. 116, 68305 Mannheim, Germany; Catalogue No. 1814443) was used for these transfections. Every fifth well containing HuH-7 blue cells was not treated. Stock solutions with a concentration of 20 μmol/l were made from the HCV 1-2, GAL1-2, and K22 dsRNAs. 1.6 μl of this stock solution was in each case mixed with 0.9 μl Fugene 6 and 108 μl DMEM. The dsRNA was therefore present at a concentration of 15 nmol/l. Each of 5 wells of a 96-well cell culture plate was transfected with 20 μl of this assay. Stock solutions were made with each of the antisense DNA oligonucleotides HCVPTO1, HCVPTO2, and HCVPTO3, and a concentration of 100 μmol/l. 1.2 μl of this stock solution was in each case mixed with 2.4 μl Fugene 6 and 108 μl DMEM. The dsRNA was therefore present in a concentration of 200 nmol/l. Each of 5 wells of a 96-well cell culture plate was transfected with 20 μl of this mixture.
Detection Methods
The effect of dsRNA oligonucleotides and antisense DNA oligonucleotides on the expression of reporter genes was determined by quantifying the β-galactosidase activity by means of chemoluminescence. For this, lysates were made using the Tropix Lysebuffer (Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif. 944404; Catalogue No. BD100LP) in accordance with manufacturer's instructions. Chemoluminescence measurements were quantified as follows:
5 μl of lysate were placed in each reagent vessel and filled to 30 μl with β-Gal assay buffer (1 ml 1 mol/l sodium phosphate buffer, pH 8.0; 10 μl 1 mol/l MgCl2, 10 μl 1.25 mg/ml Galakton [Tropix GC020, Applied Biosystems]; 9 ml deiodized water). Ml β-Gal stop mix (1 ml 2 mol/l NaOH, 250 μl 2.5% Emerald Enhancer [Applied Biosystems, Tropix, LAY250], 8.75 ml deionized water), mixed thoroughly, and immediately measured in the luminometer. If not otherwise noted, all reagents were supplied by SIGMA. Luminescence was measured in each case using the Berthold Sirius luminometer (Berthold Detection Systems GmbH, Bleichstr. 56-58, 75173 Pforzheim, Germany). 5 wells of a 96-well cell culture plate were analyzed per transfection assay. β-galactosidase activity was determined in each case, and the average of the 5 individual values was established. The average value for untransfected cells is arbitrarily defined as 1.0. The average values for transfected cells are then expressed as a ratio with the average for untransfected cells. For example, a value of 0.6 corresponds to an inhibition of β-galactosidase activity by 40% in comparison to untreated cells. The results are shown in FIG. 10.
Results
With transfection of sequence-specific antisense oligonucleotides (200 nmol/l) and dsRNA oligonucleotides (50 nmol/l) in the HuH-7 blue cell line, a reduction in β-galactosidase activity was detectable. HCVPTO1 reduced the activity of β-galactosidase by 35%, and HCVPTO2 by 40%. The HCVPTO3 oligonucleotide used as the negative control increased the activity by 40% as compared to untreated cells. The HCV1-2 and GAL1-2 dsRNAs inhibited β-galactosidase activity with comparable effectiveness. β-galactosidase activity decreased by 37% in each case, as compared with untreated cells. The K22 nonspecific control increased activity by 15% in comparison with untreated cells.
Example 2
Treatment of a HCV Infected Patient with dsRNA
In this Example, HCV specific double stranded dsRNAs are injected into HCV infected patients and shown to specifically inhibit HCV gene expression.
dsRNA Administration and Dosage
The present example provides for pharmaceutical compositions for the treatment of human HCV infected patients comprising a therapeutically effective amount of a HCV specific dsRNA as disclosed herein, in combination with a pharmaceutically acceptable carrier or excipient. DsRNAs useful according to the invention may be formulated for oral or parenteral administration. The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes, among others. One of skill in the art can readily prepare dsRNAs for injection using such carriers that include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Additional examples of suitable carriers are found in standard pharmaceutical texts, e.g. “Remington's Pharmaceutical Sciences”, 16th edition, Mack Publishing Company, Easton, Pa., 1980.
Example 3
RNA Purification and Analysis
Efficacy of the dsRNA treatment is determined at defined intervals after the initiation of treatment using real time PCR on total RNA extracted from peripheral blood. Cytoplasmic RNA from whole blood, taken prior to and during treatment, is purified with the help of the RNeasy Kit (Qiagen, Hilden) and HCV mRNA levels are quantitated by real time RT-PCR.as described previously (Eder, M., et al., Leukemia(1999) 13:1383-1389; Scherr M et al., BioTechniques. (2001) 31:520-526).
Example 4
HCV-specific dsRNA Expression Vectors
HCV-specific dsRNA molecules that interact with HCV target RNA molecules and modulate HCV gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, for example, Couture et A, 1996, TIG., 12, 5 1 0, Skillern et A, International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann et al., 1995, Proc. Natl. Acad. Sci. USA 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and cotransfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, the dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are preferably DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka et al. (1992, Curr. Topics in Micro.and Immunol. 158:97-129)), adenovirus (see, for example, Berkner et al. (1988, BioTechniques 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992, Cell 68:143-155)), or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al., 1985, Science 230:1395-1398; Danos and Mulligan, 1988, Proc. NatI. Acad. Sci. USA 85:6460-6464; Wilson et al., 1988, Proc. NatI. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the liver e.g albumin regulatory sequence (Pinkert et al., 1987, Genes Dev. 1:268276).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Preferably, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
DsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single target gene or multiple target genes over a period of a week or more are also contemplated by the present invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection. of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The nucleic acid molecules of the invention described above can also be generally inserted into vectors and used as gene therapy vectors for human patients infected with HCV. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
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10384512
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alnylam europe ag
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/44
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Mar 27th, 2007 12:00AM
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Jan 22nd, 2003 12:00AM
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https://www.uspto.gov?id=US07196184-20070327
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Double-stranded RNA (DSRNA) and method of use for inhibiting expression of the AML-1/MTG8 fusion gene
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The present invention relates to the specific inhibition of expression of a fusion gene in mammals using a short double stranded RNA. The dsRNA is approximately 19–24 nucleotides in length, and has a nucleotide sequence which is complementary to at least a part of the target gene. The dsRNAs of the present invention are useful for treating diseases caused by chromosomal aberrations, particularly malignant diseases such as lymphoma and leukemia.
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7196184
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1. An isolated double stranded ribonucleic add molecule (dsRNA) comprising two separate non-linked RNA strands, a strand S1 and a complementary RNA strand, wherein said S1 strand and said complementary strand consists of from 20 to less than 30 nucleotides in length and said S1 strand being complementary to a region of a target fusion gene comprising the fusion site in said target fusion gene, and wherein said target fusion gene is created by the fusion of two genes in a cell as a result of chromosomnal aberration wherein the S1 strand consists of the sequence SEQ ID NO:1 and said complementary RNA strand consists of the sequence SEQ ID NO: 2.
2. A dsRNA according to claim 1, wherein at least one end of the dsRNA is modified, in order to counteract degradation in the cell or dissociation into the individual strands.
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2
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RELATED APPLICATIONS
This application claims priority under 35 U.S.C. § 119 to German Application No. DE 102 02 419.7, which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
Chromosomal aberrations play a central role in the pathogenesis of many human malignant diseases, including hematologic neoplasms such as lymphoma and leukemia. Chromosomal abnormalities, characterized by structural changes or defects in one or more chromosomes, generally involve translocation, wherein a chromosome fragment is switched between non-homologous chromosomes; inversion, wherein the nucleotide sequence of a chromosome fragment is reversed; deletion (loss of a chromosomal fragment); insertion (incorporation of genetic material); duplication (repetition of an individual chromosome segment); or ring formation. These acquired genetic anomalies usually result in either activation of a quiescent gene or creation of a hybrid gene encoding a chimeric fusion oncoprotein, which triggers the malignant transformation. The chimeric fusion proteins created by cancer-associated chromosomal anomalies are ideal therapeutic targets because they are unique to the disease; they only exist in the malignant cells, not in the patient's normal cells (Cobaleda, C. et al., Bioassays (1995) 23:922).
A number of therapeutic agents which target expression of chimeric fusion genes are known in the art, including zinc-finger proteins (Choo, Y., et al., Nature (1994) 372:642), hammerhead-based ribozymes (James, H. A, and I. Gibson, Blood (1998) 91:371), and antisense RNA (Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994) 91:4504–4508). Each of these agents have inherent limitations. Zinc-finger proteins act at the DNA level, interacting with the target sequence and blocking transcription. However, gene fusions occur randomly and within introns, hence requiring a unique or “custom” zinc-finger for each patient. Antisense approaches, using either single-stranded RNA or DNA, act in a 1:1 stoichiometric relationship and thus have low efficacy, as well as questionable specificity (Skorski et al., supra). Hammerhead ribozymes, which because of their catalytic activity can degrade a higher number of target molecules, have been used to overcome the stoichiometry problem associated with antisense RNA. However, hammerhead ribozymes require specific nucleotide sequences in the target gene, which are not always present.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M. , et al., Nature (2000) 404:293–296). In other words, RNAi involves a catalytic-type reaction whereby new siRNAs are generated through successive cleavage of long dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi. WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. Sharp, P. A. , Genes & Dev. (2001) 15:485–490, suggests that dsRNA from a related but not identical gene (i.e.,>90% homologous) can be used for gene silencing if the dsRNA and target gene share segments of identical and uninterrupted senquences of significant length, i.e., more than 30–35 nucleotides. Unfortunately, the use of long dsRNAs in mammalian cells to elicit RNAi is usually not practical, due to the deleterious effects of the interferon response, as well as the inherent difficulties in delivering large molecules into a cell.
WO 00/44895 (Limmer, 2000) discloses the use of short dsRNA of less than 25 nucleotides (siRNA) for inhibiting expression of target genes in vertebrate cells. Similarly, WO 01/75164 A2 (Tuschl et al., 2001) discloses dsRNA of about 21 to 23 nucleotides for use in gene silencing by RNAi. Although the dsRNAs described in these references are small enough for intracellular delivery, neither reference suggests the use of siRNAs for inhibiting the expression of a chimeric fusion gene. Moreover, given the fact that chimeric fusion genes contain sequences from the cellular genes from which they originate, one would anticipate problems with specificity of inhibition, i.e., inhibition of both the chimeric fusion gene and the cellular genes. According to Sijen, T., et al., Cell (2001) 107:465–476, and Lipardi, C., et al., Cell (2001) 107:297–307, one strand of the siRNA would be elongated into a region that is complementary to the cellular genes. The new siRNAs formed by subsequent cleavage of the elongated products would have sequences that correspond exclusively to the cellular gene. Thus, one would anticipate inhibition of expression of the target gene as well as the cellular genes.
Finally, Cobaleda, I. and I. Sanchez-Garcia, Blood (2000) 95(3):731–737, discloses the use of a sequence-specific catalytic RNA subunit of RNase P from E. coli (MI RNA) to cleave target mRNA corresponding to the junction site in a bcr-abl fusion gene. However, the MI RNA approach suffers from the same deficiencies as the antisense approach, namely the potential for an interferon response and the inherent difficulties in delivering large molecules to cells. Moreover, because of its large size, production of therapeutic or commercial amounts of MI RNA cannot reasonably be accomplished using solid-phase synthesis. Instead, MI RNA must be prepared through enzymatic synthesis, which is costly.
Thus, despite significant advances in the field, there remains a need for agents that target expression of chimeric fusion genes associated with chromosomal aberrations. In particular, agents that are small enough for efficient intracellular delivery, and which have both high efficacy (hence are effective at low dosages) and high specificity for the target fusion gene would be therapeutically beneficial. Such agents would be useful for treating diseases caused by chromosomal anomalies, particularly malignant diseases such as lymphoma and leukemia.
SUMMARY OF THE INVENTION
The present invention discloses a short double stranded RNA (dsRNA) that specifically inhibits the expression of fusion genes in mammals. The dsRNA may be approximately 19–24 nucleotides in length and have a nucleotide sequence that is complementary to at least a part of the target gene that contains a fusion site.
In one aspect, the dsRNA of the invention contains a first complementary RNA strand and a second RNA strand. The first complementary RNA strand has a corresponding nucleotide sequence of between about 20–23 nucleotides which is complementary to an mRNA transcript of a portion of the target gene containing a fusion site. The first complementary RNA strand and the second RNA strand of the dsRNA both have a 3′-terminus and a 5′-terminus. The nucleotide sequence of the dsRNA may be about 22 nucleotides in length. The nucleotide sequence of the dsRNA may also contain at least two nucleotides on each side of the fusion site that are complementary to the corresponding nucleotides on either side of the fusion site of the target gene. At least one of the RNA strands of the dsRNA may have a nucleotide overhang of between about one and about four nucleotides in length. The nucleotide overhang may be one or two nucleotides in length. At least one of the RNA strands of the dsRNA may have a nucleotide overhang on the 3′-terminus. Only one of the RNA strands may have a nucleotide overhang, and the overhang may be on the 3′-terminus of the first complementary RNA strand. At least one of the ends of the dsRNA may also have a linker between the first complementary RNA strand and the second RNA strand. The linker may be a chemical linker such as a hexaethylene glycol linker that links the 5′-terminus of the first complementary RNA strand with the 3′-terminus of the second RNA strand.
The nucleotide sequence on the complementary RNA strand may have at least three nucleotides on each side of the corresponding fusion site, which specifically correspond to the fusion site of the target gene. The target gene may also have at least three nucleotides on each side of the fusion site. At least one of the two RNA strands of the dsRNA may have a nucleotide overhang of between one and four nucleotides, preferably one or two nucleotides, in length. The dsRNA may have only one nucleotide overhang, preferably on the 3′-terminus of the complementary RNA strand. At least one of the ends of the dsRNA may comprise a chemical linker, such as a hexaethylene glycol linker. The linker may connect the 5′-terminus of the first complementary RNA strand and the 3′-terminus of the second RNA strand. The target gene may comprise an AML-1IMTG8 fusion site. The first complementary RINA strand of the dsRNA may have the nucleotide sequence of SEQ.ID NO:1; and the second RNA strand may have the nucleotide sequence of SEQ.ID NO:2. Alternatively, the target gene may comprise a bcr/abl fusion site, or any other known fusion site which results from a chromosomal aberration. The dsRNA may comprise a single-self complementary RNA strand, wherein one end comprises a loop structure and the other end comprises the two termini. The dsRNA may have a nucleotide overhang of between about one and about four nucleotides, preferably one or two nucleotides, in length.
In one aspect of the invention, the target gene comprises an AML-1/MTG8 fusion site. The first complementary RNA strand may have a nucleotide sequence of SEQ. ID NO:1 and the second RNA strand may have a nucleotide sequence of SEQ. ID NO:2.
In another aspect of the invention, the ribonucleic acid (RNA) may have a double stranded structure that comprises a single-self complementary RNA strand having a nucleotide sequence of between about 19 and about 24 nucleotides in length which is substantially identical to at least a part of a target gene with a fusion site in a mammalian cell. The dsRNA may contain a 3′-terminus and a 5′-terminus and the double stranded structure may contain a first end and a second end. The first end comprises a loop structure and the second end comprises the 3′-terminus and the 5′-terminus and a nucleotide overhang of between about one and about four nucleotides in length. In a further aspect, the overhang may be one or two nucleotides in length be located on the 3′ terminus.
In another aspect, the invention relates to a method for inhibiting the expression of a target gene in a mammalian cell. The method involves introducing a dsRNA of the invention into a cell. The dsRNA comprises a double stranded structure having a nucleotide sequence of between about 19 and about 24 nucleotides in length which is substantially identical to at least a part of a target gene with a fusion site in the mammalian cell. The method further involves maintaining the cell under conditions and for a time sufficient to obtain degradation of mkNA of the target gene and inhibition of the expression of the target gene in the cell.
In a further aspect, the invention provides for a method of inhibiting the expression of a target gene in a mammalian cell using a dsRNA comprising a first complementary RNA strand, containing a corresponding nucleotide sequence which is complementary to an niRNA transcript of a portion of the target gene, and a second RNA strand. The first complementary RNA strand and the second RNA strand comprise a 3′-terminus and a 5′-terminus and may have a nucleotide sequence of between about 20 and about 23 nucleotides in length. In one embodiment, the nucleotide sequence may be about 22 nucleotides in length. In another embodiment, the nucleotide sequence comprises at least two nucleotides on each side of a corresponding fusion site and the fusion site is complementary to the corresponding fusion site of the target gene. The nucleotide sequence of the RNA may also comprise at least three nucleotides on each side of the corresponding fusion site. In one embodiment, at least one of said RNA strands comprises a nucleotide overhang of between about one and about four nucleotides in length. In one embodiment, the nucleotide overhang is one or two nucleotides in length. In one embodiment, at least one of the RNA strands has a nucleotide overhang on the 3′-terminus. In one embodiment, only the first complementary RNA strand has a nucleotide overhang, and the overhang is on the 3′-terminus of the first complementary RNA strand. At least one of the ends of the dsRNA may comprise a chemical linker, such as a hexaethylene glycol linker. The linker may connect the 5′-terminus of the first complementary RNA strand and the 3′-terminus of the second RNA strand. The target gene may comprise an AML-1IMTG8 fusion site. The first complementary RNA strand of the dsRNA may have the nucleotide sequence of SEQ. ID NO:1; and the second RNA strand may have the nucleotide sequence of SEQ. ID NO:2. Alternatively, the target gene may comprise a bcr/abl fusion site, or any other known fusion site which results from a chromosomal aberration, including BCL-1IgH, TAL-1/TCR, TAL-1/SIL, c-MYC/IgH, c-MYC/IgL, MUM1/RF4, MUM1/IgH, RAX-5/BSAP, MLL/HRX, E2AIPBX, E2AIHLF, NPM/ALK, and NPM/MLF1. The mammalian cell may be a leukocyte or a myelogenic cell. The target gene may result from a chromosomal aberration. In one embodiment, the target gene causes or is likely to cause disease. The dsRNAs of the present invention are useful for treating diseases caused by chromosomal aberrations, particularly malignant diseases such as lymphoma and leukemia. The RNA may be produced by chemical synthesis or by an expression vector in the cell. In one embodiment, the nucleotide sequence has at least 90% identity with a part of the target gene.
In another aspect, the invention provides for a method for treating a mammal having a disease caused by the expression of a fusion gene which results from a chromosomal aberration, by administering to the mammal an RNA that inhibits the expression of the target gene. The RNA comprises a double stranded structure having a nucleotide sequence which is substantially identical to at least a part of a target gene and a nucleotide sequence of between about 19 and about 24 nucleotides in length. In one embodiment, the RNA comprises a first complementary RNA strand and a second RNA strand and the first complementary RNA strand comprises a corresponding nucleotide sequence which is complementary to an mRNA transcript of a portion of the target gene, and the first complementary RNA strand and the second RNA strand comprise a 3′-terminus and a 5′-terminus. In another embodiment, the nucleotide sequence may be between about 20 and about 23 nucleotides in length. In another embodiment, the target gene may comprise a fusion site, and the nucleotide sequence of the dsRNA may comprise at least two nucleotides on each side of the fusion site within the target gene. In another embodiment, at least one of said RNA strands may have a nucleotide overhang of between about one and about four nucleotides in length. In another embodiment, the first complementary RNA strand has a nucleotide overhang on the 3′-terminus. In another embodiment, at least one of the ends may have a linker such as hexaethylene glycol between the first complementary RNA strand and the second RNA strand. In a further aspect, the target gene may be an AML/MTG8 fusion gene, a BCR/ABL fusion gene or a target gene selected from the group of fusion genes consisting of BCL-1/IgH, TAL-1/TCR, TAL-1/SIL, c-MYC/IgH, c-MYC/JgL, MUM1/RF4, MUM1/IgH, RAX-5/BSAP, MLL/HRX, E2A/PBX, E2A/HLF, NPM/ALK, and NPM/MLF1. The target gene may be a result of a chromosomal aberration which causes or is likely to cause a disease for example, acute myelogenous leukemia.
In another aspect, the invention provides for a method of using an RNA to inhibit the expression of a target gene with a fusion site in a mammalian cell. According to the invention, the RNA may be about 19 to about 24 nucleotides in length and comprise a double stranded structure having a nucleotide sequence which is substantially identical to at least a part of a target gene with a fusion site in the mammalian cell.
In a further aspect, the invention relates to a pharmaceutical composition comprising the dsRNA of the invention and a pharmaceutically acceptable carrier. The dsRNA may have a double stranded structure and a nucleotide sequence, of between about 19 and about 24 nucleotides in length, which is substantially identical to at least a part of a target gene with a fusion site in the mammalian cell. In another aspe, the mammalian cell may be a leukocyte or a myelogenic cell. The target gene may be a result of a chromosomal aberration which causes or is likely to cause a disease such as leukemia or lymphoma.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an autoradiograph of RNase protection assays of cellular RNAs performed 20 hours after electroporation of Kasumi-1 cells with 200 nM siRNA. Protected fragments corresponding to AML-1/MTG8 having 240 nucleotides in length and to AML-1 having 100 nucleotides in length are indicated on the left. The electroporated siRNAs are indicated at the top of Lanes 1 to 5. Lane 6 shows a transfer RNA (tRNA) control to monitor completeness of digestion; Lane 7 represents the undigested 315-nucleotide probe.
FIG. 2 is a graphic representation of the ratios between AML-1/MTG8 and AML-1 intensities. Band intensities from the experiment in FIG. 1 were quantified by phosphoimaging and the relative ratios of AML-1/MRG8 mRNA to AML-1 mRNA was determined. Electroporated siRNAs are indicated at the bottom.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the specific inhibition of expression of a fusion gene in a mammal using a short double stranded RNA (dsRNA). dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates. Using a mammalian tissue cell culture, the present inventors have demonstrated that dsRNA of approximately 19–24 nucleotides, preferably 20–23 nucleotides, and most preferably 22 nucleotides in length, which have a nucleotide sequence complementary to a target fusion gene, can specifically and efficiently mediate RNAi. The present invention encompasses these short dsRNAs and their use for specifically inactivating gene function. The use of these dsRNAs enables the targeting of mRNAs of fusion genes resulting from a chromosomal aberration. Thus, the dsRNAs of the present invention are useful for treating diseases caused by chromosomal aberrations, particularly malignant diseases such as lymphoma and leukemia.
The dsRNAs of the present invention comprise a double stranded structure, and have a nucleotide sequence which is substantially identical to at least a part of the target gene. “Identity,” as known in the art, is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, .e.g, Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W. , ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SJAM.JJ Applied Math. (1988) 48:1073. “Substantially identical,” as used herein, means there is a very high degree of homology (preferably 100% sequence identity) between the inhibitory dsRNA and the corresponding part of the target gene. However, dsRNA having greater than 90% or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. Although 100% identity is preferred, the dsRiNA may contain single or multiple base-pair random mismatches between the RNA and the target gene, provided that the mismatches occur at a distance of at least three nucleotides from the fusion site.
As used herein, “target gene” refers to a section of a DNA strand of a double-stranded DNA that is complementary to a section of a DNA strand, including all transcribed regions, that serves as a template for transcription. The target gene is therefore usually the sense strand. As used herein, “gene” refers to a region in DNA, bounded by an initiation (start) site and termination site, that is transcribed into a single primary transcript. As used herein, “cellular gene” refers to a gene present in a cell or organism.
The term “complementary RNA strand” refers to the strand of the dsRNA which is complementary to an mRNA transcript that is formed during expression of the target gene, or its processing products. “dsRNA” refers to a ribonucleic acid molecule having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. Not all nucleotides of a dsRNA must exhibit Watson-Crick base pairs. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA.
As used herein, “fusion site” refers to a site on a target gene where at least two cellular genes, which are normally present at distinct locations on the chromosome or on different chromosomes, are joined as a result of a chromosomal aberration. “Chromosomal aberrations” or “chromosomal abnormalities,” which are characterized by structural changes or defects in one or more chromosomes, generally involve translocation, wherein a chromosome fragment is switched between non-homologous chromosomes. Chromosomal aberration can also be caused by other acquired genetic alterations, including inversion (wherein the nucleotide sequence of a chromosome fragment is reversed), deletion (loss of a chromosomal fragment), insertion (incorporation of genetic material), duplication (repetition of an individual chromosome segment), and ring formation.
The term “corresponding fusion site,” as used herein, refers to a fusion site in the nucleotide sequence of an RNA strand of the dsRNA that is complementary to the fusion site of the target gene. A dsRNA “comprises” a fusion site when at least one nucleotide is present on one side of the fusion site. The remainder of the complementary strand comprises nucleotides on the opposite side of the corresponding fusion site. Thus, the fusion site is not located entirely at the beginning or end of the complementary segment of the RNA strand. The complementary segment of the RNA strand preferably comprises at least 16 nucleotides. “Introducing into” means uptake or absorption in the cell, as is understood by those skilled in the art. Absorption or uptake can occur through cellular processes, or by auxiliary agents or devices.
In one embodiment, the invention relates to an RNA having a double-stranded structure and a nucleotide sequence which is substantially identical to at least a part of the target gene, which comprises the fusion site. The RNA is between about 19 and about 24 nucleotides in length. The dsRNA comprises two complementary RNA strands, one of which comprises a nucleotide sequence which is substantially identical to a portion of the target gene. Preferably, the nucleotide sequence of the RNA which contains the corresponding fusion site has at least three nucleotides on each side of the fusion site. For example, a sequence of 21 nucleotides in length would have at least three nucleotides on one side of the corresponding fusion site, and up to 18 nucleotides on the opposite side of the fusion site. Surprisingly, the present inventors have discovered that dsRNAs having this nucleotide configuration demonstrate exceptional efficiency and specificity of activity.
In a preferred embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of between one and four, preferably one or two nucleotides. As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure when the 5′-terminal end of one RNA strand extends beyond the 3′-terminus end of the other strand, or vice versa. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of one nucleotide overhang strengthens the interference activity of dsRNA, without diminishing the overall stability of the structure, as typically happens with dsRNA having nucleotide overhangs at both ends. dsRNA having only one overhang has proven particularly stable and effective in a variety of cells and cell culture mediums, as well as in blood and serum. Preferably, the single-stranded overhang is located at the 3′-terminal end of the complementary RNA strand (also referred to herein as the “S1” strand). Such a configuration produces a further increase in efficiency.
The nucleotide sequence on the complementary RNA strand (S1 strand) preferably has between 20 and 23 nucleotides, most preferably 22 nucleotides. Such dsRNA are particularly robust gene silencers. The complementary RNA strand of the dsRNA strand preferably has fewer than 30 nucleotides, more preferably fewer than 25 nucleotides, and most preferably 21 to 24 nucleotides. Such dsRNA exhibit superior intracellular stability.
At least one end of the dsRNA may be modified to improve resistance to degradation and/or dissociation of the two strands of the duplex. Furthermore, the cohesiveness of the double-stranded structure formed by base pairing between the complementary RNA strands can be further improved by the presence of one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, including through covalent, ionic or hydrogen bonds; hydrophobic interactions, preferably van der Waals or stacking interactions; or by means of metal-ion coordination. The purines of the dsRNA may also be replaced with purine analogues. Most preferably, the chemical linkage is achieved using a hexa-ethylene glycol linker on one end of the dsRNA. In a preferred embodiment, the linkage is formed between the 5′-terminus of the complementary RNA strand and the 3′-terminus of the second RNA strand.
In another embodiment, the present invention relates to a method for inhibiting the expression of a target gene comprising a fusion site using a dsRNA. The method comprises introducing a dsRNA having a nucleotide sequence which is substantially identical to at least a part of a target gene into a mammalian cell. The RNA is preferably between 20 and 23 nucleotides in length, most preferably 22 nucleotides. The resulting cell is maintained under conditions and for a time sufficient to achieve degradation of mRNA of the target gene, thereby silencing expression of the target gene.
In still another embodiment, the invention relates to a method for treating a mammal having a disease caused by the expression of a fusion gene which results from a chromosomal aberration. The method comprising administering the dsRNA of the invention to the animal, such that expression of the target fusion gene is silenced. Because of their surprisingly improved specificity, the dsRNAs of the present invention specifically target mRNAs of chimeric fusion genes of diseased cells and tissues, without affecting the surrounding normal cells. Thus, the dsRNAs of the present invention are particularly useful for treating diseases caused by chromosomal aberrations, particularly malignant diseases such as lymphoma and leukemia.
Examples of diseases which can be treated using the dsRNA of the invention include, without limitation, acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML), mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, T-cell acute lymphoblastic leukemia, Burkett lymphoma, myeloma, immunocytoma, acute promyelocytic leukemia, chronic myeloid/acute lymphoblastic leukemia, acute leukemia, B-cell acute lymphoblastic leukemia, anaplastic large cell lymphoma, and myelodysplastic syndrome/acute mycloid leukemia. These leukemias and lymphomas can be treating using a dsRNA specifically designed to inhibit expression of the aberrant fusion gene. Although the present examples describe the preparation of dsRNAs which target the AML-1/MTG8 and bcr/abl fusion genes, other dsRNAs can be constructed to target other fusion genes using well-known techniques and by following the teachings of the present invention. Examples of other fusion genes which can be used in the methods of the invention include, without limitation, BCL-1/IgH, TAL-1/TCR, TAL-1/SIL, c-MYC/IgH, c-MYC/IgL, MUM1/RF4, MUM1/IgH, RAX-5/BSAP, MLL/HRX, E2A/PBX, E2A/HLF, NPM/ALK, and NPM/MLF 1.
Acute myelogenous leukemias (AML) are heterogeneous, malignant diseases of the hemopoietic system. AML is caused by expression of an aberrant fusion gene, which results in loss of the ability of the cell to differentiate, while retaining the potential to proliferate. This leads to the promulgation of a malignant cell clone, with resultant suppression of normal hematopoiesis. Untreated, AML causes death, usually within a few weeks. The incidence of AML is age-dependent, rising from 1/100,000 in persons under 30 years of age to 14/100,000 in persons over 70.
As many as 90% of cases of adult AML demonstrate chromosomal aberrations. One of the most frequent aberrations is the t(8;21) (q22;q22) translocation, which occurs in 10–15% of all AML cases. In this translocation, the AML-1 transcription factor, which is essential for hematopoiesis, is fused with the MTG8 transcription repressor. The resulting fusion protein (AML-1/MTG8) contains almost the entire MTG8 sequence instead of the C-terminal transactivation domain of AML-1. Expression of this faulty gene results in inhibition of cell differentiation in CD34-positive cells, as well as initiation of leukemic transformation in the affected cells.
In an exemplified embodiment, the target gene comprises an AML-1/TG8 fusion gene. In this example, the complementary RNA (S1) strand of the dsRNA has the sequence set forth in SEQ ID NO:1, and the second (S2) strand has the sequence of SEQ ID NO:2. Such a construct is useful for treating either acute myelogenic leukemia or chronic myelogenic leukemia. As described in more detail below, the dsRNA can be administered using any acceptable carrier, including buffer solutions, liposomes, micellar structures, and capsids, the latter two of which facilitate intracellular uptake of dsRNA. Although the therapeutic agent can be administered by a variety of well known techniques, again as discussed below, presently preferred routes of administration include inhalation, oral ingestion, and injection, particularly intravenous or intraperitoneal injection, or injection directly into the affected bone marrow. An example of a preparation suitable for inhalation or injection is a simple solution comprising the dsRNA and a physiologically tolerated buffer, particularly a phosphate buffered saline solution.
In yet another embodiment, the invention relates to a pharmaceutical compostion for treating a disease caused by a chromosomal aberration. In this aspect of the invention, the dsRNA of the invention is formulated as described below. The pharmaceutical composition is administered in a dosage sufficient to inhibit expression of the target gene. The present inventors have found that compositions comprising the dsRNA can be administered at a unexpectedly low dosages. Surprisingly, a dosage of 5 mg dsRNA per kilogram body weight per day is sufficient to inhibit or completely suppress expression of the target gene. Furthermore, the pharmaceutical composition is highly specific in inhibiting expression of the target gene, without affecting expression of the individual cellular genes from which the fusion gene originated. Because of the high specificity of these dsRNA and low dosage requirements, side effects are either minimal or nonexistent.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease of disorder is the amount necessary to effect that at least 25% reduction.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
The dsRNAs encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
For oral administration, the dsRNAs useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavouring agents, colouring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, the daRNAs of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The dsRNAS useful according to the invention may also be presented as liposome formulations.
In general a suitable dose will be in the range of 0.01 to 100 mg per kilogram body weight of the recipient per day, preferably in the range of 0.2 to 10 mg per kilogram body weight per day, and most preferably about 5 mg per kilogram body weight per day. The desired dose is preferably presented once daily, but may be dosed as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. These sub-doses may be administered in unit dosage forms, for example, containing 10 to 1500 mg, preferably 20 to 1000 mg, and most preferably 50 to 700 mg of active ingredient per unit dosage form.
Dosages useful according to the invention will vary depending upon the condition to be treated or prevented and on the identity of the inhibitor being used. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made on the basis of in vivo testing using an animal model, such as a mouse model for lematological malignancies.
Advances in mouse genetics have generated a number of mouse models for the study of hematopoietic malignancies including leukemias, lymphomas and acute myelogenous leukemia. The MMHCC (Mouse models of Human Cancer Consortium) web page (emice.nci.nih.gov), sponsored by the National Cancer Institute, provides disease-site-specific compendium of known cancer models, and has links to the searchable Cancer Models Database (cancermodels.nci.nih.gov) as well as the NCI-MMHCC mouse repository. Examples of the genetic tools that are currently available for the modeling of leukemia and lymphomas in mice, and which are useful in practicing the present invention, are described in the following references: Bernardi, R., et al. (2002), “Modelling haematopoietic malignancies in the mouse and clinical implications,” Oncogene 21, 3445–3458; Maru, Y. (2001), Molecular biology of chronic myeloid leukemia, Int. J. Hematol., 73, 308–322; Pandolfi, P. P. (2001), In vivo analysis of the molecular genetics of acute promyelocytic leukemia, Oncogene 20, 5726–5735; Pollock, J. L., et al. (2001) Mouse models of acute promyelocytic leukemia, Curr. Opin. Hematol. 8, 206–211; Rego, E. M., et al. (2001) Analysis of the molecular genetics of acute promyelocytic leukemia in mouse models, Semin. in Hemat. 38, 54–70; Shannon, K. M., et al. (2001) Modeling myeloid leukemia tumors suppressor gene inactivation in the mouse, Semin. Cancer Biol. 11, 191–200; Van Etten, R. A., (2001) Pathogenesis and treatment of Ph+ leukemia: recent insights from mouse models, Curr. Opin. Hematol. 8, 224–230; Wong, S., et al. (2001) Modeling Philadelphia chromosome positive leukemias, Oncogene 20, 5644–5659; Higuchi M et al. (2002) Expression of a conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of human t(8;21) acute myeloid leukemia, Cancer Cell 1(1):63–74; Bichi, R. et al. (2002) Human chronic lymphocytic leukemia modeled in mouse by targeted TCL1 expression, Proc. Natl. Acad. Sci. USA, Vol. 99, Issue 10, 6955–6960; Phillips J A. Et al. (1992) The NZB mouse as a model for chronic lymphocytic leukemia, Cancer Res. 52(2):437–43; Harris A W et al. (1988) The E mu-myc transgenic mouse. A model for high-incidence spontaneous lymphoma and leukemia of early B cells, J Exp Med. 167(2):353–71; Zeng X X et al. (1998) The fetal origin of B-precursor leukemia in the E-mu-ret mouse, Blood. 92(10):3529–36; Eriksson B et al. (1999) Establishment and characterization of a mouse strain (TLL) that spontaneously develops T-cell lymphomas/leukemia, Exp Hematol. 27(4):682–8; and Kovalchuk A. et al. (2000) Burkitt lymphoma in the mouse, J Exp Med.192(8):1183–90. Mouse repositories can also be found at: The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Network and at the European Mouse Mutant Archive.
In addition to their administration singly, the dsRNAs useful according to the invention can be administered in combination with other known agents effective in treatment of malignant diseases. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
The present invention is illustrated by the following examples, which are not intended to be limiting in any way.
EXAMPLES
Example 1
Inhibition of AML-1/MTG8 Fusion Gene Expression by RNA Interference
In this Example, AML-1/MTG8 double stranded siRNAs transfected into Kasumi-1 tissue culture cells are shown to specifically inhibit AML-1/MTG8 fusion gene expression.
Synthesis and Preparation of dsRNAs
Oligoribonucleotides were synthesized with an RNA synthesizer (Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and purified by High Pressure Liquid Chromatography (HPLC) using NucleoPac PA-100 columns, 9×250 mm(Dionex Corp.; low salt buffer: 20 mM tris, 10 mM NaClO4, pH 6.8, 10% acetonitrile; flow rate: 3 ml/min). Formation of double stranded siRNAs was then achieved by heating a stoichiometric mixture of the individual complementary strands (10 M) to 95° C. for 5 minutes in 25 mM tris-HCl, pH 7.5, and 100 mM NaCl, followed by subsequent cooling for 30 minutes to 37° C.
dsRNA molecules with linkers were produced by solid phase synthesis and addition of hexaethylene glycol as a non-nucleotide linker (D. Jeremy Williams, Kathleen B. Hall, Biochemistry, 1996, 35, 14665–14670). A Hexaethylene glycol linker phosphoramidite (Chruachem Ltd, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, G20 OUA, Scotland, UK) was coupled to the support bound oligoribonucleotide employing the same synthetic cycle as for standard nucleoside phosphoramidites (Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany) but with prolonged coupling times. Incorporation of linker phosphoramidite was comparable to the incorporation of nucleoside phosphoramidites.
Two dsRNAs (AGF2 and AGF-3) were generated that target the sequences immediately adjacent to the site where the AML-1 gene is fused to the MTG8 gene. K3 and HCV10L dsRNAs were used as internal controls. The sequences of the respective dsRNAs (SEQ ID Nos. 1–6) are depicted below:
AGF2 dsRNA:
S2:
5′-CCUCGAAAUCGUACUGAGAAG-3′
(SEQ ID NO:2)
S1*:
3′-UUGGAGCUUUAGCAUGACUCUUC-5′
(SEQ ID NO:1)
The S1 strand is complementary to the coding strand of the AML-1/MTG8 fusion gene. Underlined sequences correspond to MTG8 gene sequences whereas the sequences that are not underlined correspond to AML-1 gene sequences.
AGF3L dsRNA:
AGF3L dsRNA has the same sequence as AGF2 dsRNA but, in addition, includes a hexaethylene glycol linker (*) that joins the 5′-end of the S1 strand to the 3′-end of the S2 strand. Underlined sequences correspond to MTG8 gene sequences whereas the sequences that are not underlined correspond to AML-1 gene sequences.
K3 dsRNA:the S1 strand is complementary to a sequence of the 5′-untranslated region of a neomycin resistance gene:
S2:
5′-GAUGAGGAUCGUUUCGCAUGA-3′
(SEQ ID NO:4)
S1:
3′-UCCUACUCCUAGCAAAGCGUACU-5′
(SEQ ID NO:3)
HCV10L dsRNA:the S1 strand is complementary to a sequence of the HCV gene. A hexaethylene glycol linker (*) joins the 5′-end of the S1 strand to the 3′-end of the S2 strand:
Transfection of dsRNAs into Kasumi-1 Cells
The Kasumi-1 cell line (Asou, H. et al. [1991] Blood 77, 2031–2000 36), harbors a t(8;21) translocation by which the AML-1 gene is fused to the MTG-1 gene.
The dsRNAs described above were transfected into these cells using the following protocol. DsRNAs were first added to 106 cells in 100 μl RPMI1640 with 10% FCS to a final concentration of 200 nM and then electroporated in a 0.4 cm-wide electroporation cuvette at 300 V for 10 minutes using a Fischer Electroporator (Fischer, Heidelberg). After a 15-minute incubation at room temperature, the cell suspension was transferred to 2 ml RPMI1640 with 10% fetal calf serum, and incubated a further 20 hours at 37° C., 5% CO2, and 95% humidity prior to processing and analysis.
RNA Purification and Analysis
Cytoplasmic RNA was purified with the help of the RNeasy Kit (Qiagen, Hilden) and analyzed using a RNase protection assay as previously described (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Strul, K. (1993) Current Protocols in Molecular Biology, Greene and Wiley, New York, N.Y.). The following modifications were included: the hybridization volume was reduced to 15 μl, the hybridization temperature was 60° C., and the RNase digestion was performed in a total volume of 175 μl.
SEQ ID NO: 7 represents the sequence of the RNA probe. Sequences protected by AML1l/MTG8-mRNA are highlighted, sequences protected by AML1-mRNA are underlined:
Sequence of the 100 nucleotide fragment protected by the AML-1 mRNA is:
5′-UCGAGGUUCU CGGGGCCCAU CCACUGUGAU UUUGAUGGCU CUGUGGUAGG
(SEQ ID NO:8)
UGGCGACUUG CGGUGGGUUU GUGAAGACAG UGAUGGUCAG AGUGAAGCUU-3′
Sequence of the 240 nucleotide fragment protected by the AML-1/MTG8 fusion mRNA is:
5′-AACGUUGUCG GUGUAAAUGA ACUGGUUCUU GGAGCUCCUU GAGUAGUUGG
(SEQ ID NO:9)
GGGAGGUGGC AUUGUUGGAG GAGUCAGCCU AGAUUGCGUC UUCACAUCCA
CAGGUGAGUC UGGCAUUGUG GAGUGCUUCU CAGUACGAUU UCGAGGUUCU
CGGGGCCCAU CCACUGUGAU UUUGAUGGCU CUGUGGUAGG UGGCGACUUG
CGGUGGGUUU GUGAAGACAG UGAUGGUCAG AGUGAAGCUU-3′
After RNase treatment, the RNA was analyzed by polyacrylamide gel electrophoresis under denaturing conditions (see FIG. 1) and the relative amount of different RNAase resistant fragments was quantitated by phosphor imaging. As can be seen in FIG. 1, the identity of the transfected dsRNAs is indicated above each lane. Lane 1 shows the cytoplasmic RNA from a cell that was electroporated in the absence of a dsRNA. A 315-nucleotide long RNA that is complementary to the AML-1/MTG8 fusion site was used as the undigested sample RNA (FIG. 1, Lane 7). The denaturing temperature was 95° C., the hybridization temperature 60° C. Conditions for complete digestion were tested using tRNA (FIG. 1, Lane 6).
Both AML-1/MTG8-specific fragments having a length of 240 nucleotides as well as AML-1-specific fragments having a length of 100 nucleotides were seen in all assays (see arrows, FIG. 1). Bands corresponding to 91 nucleotide long fragments result from the expression of the untranslocated wild type allele. Neither the control—nor the AML-1/MTG8-specific dsRNAs reduced the AML-1 signal of non-fused mRNA (FIG. 1, compare lane 1 with lanes 3 and 5). In contrast to the control dsRNAs (K3 and HCV10L; FIG. 1, lanes 3 and 5), both AML-1/MTG8 fusion mRNA specific dsRNAs: AGF2 dsRNA, in which both strands are non linked (see FIG. 1, lane 2) and AGF3L dsRNA (in which both strands are linked by a hexaethylene glycol linker (see FIG. 1, lane 4)), reduced the AML-1/MTG8 signal significantly. Whereas the ratio of the AML-1/MTG8 to AML-1 signal fluctuates between 1.1 and 1.4 both in cells that were electroporated in the absence of dsRNAs and in cells that were transfected with control dsRNAs, electroporation in the presence of AML-1/MTG8-specific dsRNAs resulted in a significant reduction in this ratio to between 0.4 and 0.6 (FIG. 2). Thus, AML-1/MTG8-specific dsRNAs containing a hexaethylene glycol linker can specifically reduce expression of the AML-1/MTG8 fusion gene to 46% of the expression seen in the absence of AML-1/MTG8-specific dsRNAs, whereas the expression of the untranslocated allele remained unaltered either in the presence or absence of dsRNAs. Assuming an electroporation efficiency of 50%, these results indicate that transfected AML-1/MTG8-specific dsRNAs are highly effective at specifically targeting and degrading AML-1/MTG8 fusion gene transcripts.
Example 2
Inhibition of Bcr-Abl Fusion Gene Expression by RNA Interference
In this Example, Ber-Abi-specific double stranded siRNAs transfected into CD34+primary hematopoietic cells from CML patients are shown to specifically inhibit Ber-Abl gene expression.
SiRNA Synthesis
21-nt single-stranded RNAs (BCR-ABL-1 and BCR-ABL-2) directed against the fusion sequence of bcr-abl are chemically synthesized with or without a hexaethylene glycol linker as described in Example 1.
The sense and antisense sequences of the siRNAs are:
Bcr-Abl-specific double stranded siRNAs were transfected into CD34+ primary hematopoietic cells from CML patients. Cells were purified to>95% and cultured as previously described (Scherr M. et al. Blood. 2002; 99: 709–712). Primary CD34+ are cultured in X-VIVO/1% HSA with recombinant human SCF (100 ng/ ml), Flt-3-ligand (100 ng/ml), and TPO (20 ng/ml) before electroporation, and GM-CSF and IL-3 (10 ng/ml each) is added thereafter.
The dsRNAs described above are transfected into these cells using the following protocol. DsRNAs are first added to 106 cells in 100 μl RPMI1640 with 10% FCS to a final concentration of 200 nM and then electroporated in a 0.4 cm-wide electroporation cuvette at 300 V for 10 minutes using a Fischer Electroporator (Fischer, Heidelberg).
After a 15-minute incubation at room temperature, the cell suspension was transferred into fresh media (see above) and incubated a further 20 hours at 37° C., 5% CO2, and 95% humidity prior to processing and analysis.
RNA Purification and Analysis
Cytoplasmic RNA was purified with the help of the RNeasy Kit (Qiagen, Hilden) and Bcr-abl mRNA levels were quantitated by real time RT-PCR.
Real Time PCR Analysis
Real-time Taqman-RT-PCR is performed as described previously (Eder M et al. Leukemia 1999; 13: 1383–1389; Scherr M et al. BioTechniques. 2001; 31: 520–526).
The probes and primers are:
bcrFP:
5′-AGCACGGACAGACTCATGGG-3′,
(SEQ ID NO: 15)
bcrRP:
5′-GCTGCCAGTCTCTGTCCTGC-3′,
(SEQ ID NO: 16)
bcr-Taqman-probe:
5′-AGGGCCAGGTCCAGCTGGACCC-3′
(SEQ ID NO: 17)
covering the exon b5/b6 boundary,
ablFP:
5′-GGCTGTCCTCGTCCTCCAG-3′,
(SEQ ID NO: 18)
ablRP:
5′-TCAGACCCTGAGGCTCAAAGT-3′,
(SEQ ID NO: 19)
abi-Taqman-probe:
5′-ATCTGGAAGAAGCCCTTCAGCGGC-3′
(SEQ ID NO: 20)
covering the exon 1a/6 Bcr-abl
RNA levels in primary CD34+
hematopoietic cells from CML
patients transfected with BCR-ABL
siRNAs or control siRNAs (with or
without hexaethylene glycol
linker) are determined by real
time RT-PCR and standardized
against an internal control e.g.
GAPDH mRNA levels.
Bcr-abl RNA levels in primary CD34+ hematopoietic cells from CML patients transfected with BCR-ABL siRNAs or control siRNAs (with or without hexaethylene glycol linker) are determined by real time RT-PCR and standardized against an internal control e.g. GAPDH mRNA levels.
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10349320
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alnylam europe ag
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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536/ 23.1
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Jan 6th, 2009 12:00AM
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Mar 7th, 2003 12:00AM
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https://www.uspto.gov?id=US07473525-20090106
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Compositions and methods for inhibiting expression of anti-apoptotic genes
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The present invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of an anti-apoptotic gene, comprising a complementary RNA strand having a nucleotide sequence which is less that 25 nucleotides in length and which is substantially identical to at least a part of an apoptotic gene, such as a Bcl gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of an anti-apoptotic gene using the pharmaceutical composition; and methods for inhibiting the expression of an anti-apoptotic gene in a cell.
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7473525
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1. A method for inhibiting the expression of an anti-apoptotic gene in a cell, the method comprising: (a) introducing into the cell in vitro a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA consists of less than 25 nucleotides in length and comprises a complementary RNA strand comprising a complementary nucleotide sequence which is complementary to at least a part of the Bcl-2 gene, and wherein the complementary nucleotide sequence consists of at least 24 nucleotides in length and comprises SEQ ID NO:2; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the anti-apoptotic gene, thereby inhibiting expression of the Bcl-2 gene in the cell.
2. A method for inhibiting the expression of an anti-apoptotic gene in a cell, the method comprising: (a) introducing into the cell in vitro a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA consists of less than 25 nucleotides in length and comprises a complementary RNA strand comprising a complementary nucleotide sequence which is complementary to at least a part of the Bcl-2 gene, and wherein the complementary nucleotide sequence consists of at least 24 nucleotides in length and comprises SEQ ID NO:4; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the anti-apoptotic gene, thereby inhibiting expression of the Bcl-2 gene in the cell.
3. The method of claim 1, wherein the dsRNA further comprises a sense RNA strand, and wherein at least one of the complementary RNA strand or sense RNA strand comprises a nucleotide overhang of 1 to 4 nucleotides in length, and wherein the sense RNA strand comprises the sequence of SEQ ID NO: 1.
4. The method of claim 2, wherein the dsRNA further comprises a sense RNA strand, and wherein at least one of the complementary RNA strand or sense RNA strand comprises a nucleotide overhang of 1 to 4 nucleotides in length, and wherein the sense RNA strand comprises the sequence of SEQ ID NO:3.
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4
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RELATED APPLICATIONS
This application is a continuation-in-part of International Application No. PCT/EP02/00151, which designated the United States and was filed on Jan. 9, 2002, which claims the benefit of German Patent No. 101 00 586.5, filed on Jan. 9, 2001. The entire teachings of the above application(s) are incorporated herein by reference.
FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of an anti-apoptotic target gene, such as a Bcl gene.
BACKGROUND OF THE INVENTION
Many diseases, incuding cancers, arise from the abnormal expression or activity of a particular gene, a group of genes, or a mutant form of protein. The therapeutic benefits of being able to selectively silence the expression of these genes is obvious.
A number of therapeutic agents designed to inhibit expression of a target gene have been developed, including antisense ribonucleic acid (RNA) (see, e.g., Skorski, T. et al., Proc. Natl. Acad. Sci. USA (1994) 91:4504-4508) and hammerhead-based ribozymes (see, e.g., James, H. A, and I. Gibson, Blood (1998) 91:371). However, both of these agents have inherent limitations. Antisense approaches, using either single-stranded RNA or DNA, act in a 1:1 stoichiometric relationship and thus have low efficacy (Skorski et al., supra). Hammerhead ribozymes, which because of their catalytic activity can degrade a higher number of target molecules, have been used to overcome the stoichiometry problem associated with antisense RNA. However, hammerhead ribozymes require specific nucleotide sequences in the target gene, which are not always present.
More recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of a target gene in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophilia (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kruetzer et al.).
In RNA interference, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (Hammond, S. M., et al., Nature (2000) 404:293-296). In other words, RNAi involves a catalytic-type reaction whereby new siRNAs are generated through successive cleavage of long dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence-specific degradation of endogenous messenger RNA (mRNA) through RNAi.
Gautschi et al. report that the expression levels of the anti-apoptotic proteins Bcl-1 and Bcl-xL are elevated during the development and progression of tumors (Gautschi, O., et al., J. Natl. Cancer Inst. (2001) 93:463-471). Tumor growth (but not size) was reduced by approximately 50-60% in nude mice treated with a combination of single-stranded antisense oligoribonucleotides targeted to Bcl-2 and Bcl-xL genes. However, because of the 1:1 stoichiometric relationship and thus low efficiency of antisense RNA, the anti-Bcl treatment required 20 milligrams of antisense RNA per kilogram body weight of recipient mouse per day. Producing therapeutically sufficient amounts of RNA is not only expensive, but single-stranded antisense RNA is highly susceptible to degradation by serum proteases, thus resulting in a short in vivo half-life.
Despite significant advances in the field, there remains a need for an agent that can selectively and efficiently silence a target gene using the cell's own RNAi machinery. More specifically, an agent that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target anti-apoptotic gene at a low dose, would be highly desirable. Compositions comprising such agents would be useful for treating diseases caused by the expression of these genes.
SUMMARY OF THE INVENTION
The present invention discloses double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a target gene, such as an anti-apoptotic gene, in a cell using the dsRNA. The present invention also discloses compositions and methods for treating diseases caused by the expression of a target anti-apoptotic gene (e.g., a Bcl gene). The dsRNA of the invention comprises an RNA strand (the complementary strand) having a region which is less than 25 nucleotides in length and is complementary to at least a portion of an RNA transcript of an anti-apoptotic target gene, such as Bcl-2, Bcl-w, or Bcl-xL.
In one aspect, the invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of an anti-apoptotic gene in a cell. The dsRNA comprises a complementary RNA strand having a complementary nucleotide sequence which is complementary to at least a part of the anti-apoptotic gene, and which is less than 25 nucleotides in length. The dsRNA may further comprise a sense RNA strand, and at least one of the RNA strands comprises a nucleotide overhang of 1 to 4 nucleotides, preferably 2 or 3 nucleotides in length. In a preferred embodiment, the nucleotide overhang is on the 3′-terminus of the complementary RNA strand, and the 5′-end is blunt. The complementary RNA strand and sense RNA strand have a region of complementarilty, which may be 19 to 24 nucleotides, preferably 21 to 24 nucleotides, and most preferably 22 nucleotides in length. The complementary RNA strand may be less than 30, preferably less than 25, and most preferably 21 to 24 nucleotides in length. In one embodiment, the dsRNA may have at least one, preferably two, linkers between the complementary RNA strand and the sense RNA strand, such as a chemical linker. The chemical linker may be a hexaethylene glycol linker, apoly-(oxyphosphinico-oxy-1,3-propandiol) linker, or an oligoethyleneglycol linker. The anti-apoptotic gene may be a Bcl gene, such as Bcl-2, Bcl-w, or Bcl-xL. In one embodiments, both the complementary RNA strand and the sense RNA strand comprise the sequence of SEQ ID NO:2. In another embodiment, the complementary RNA strand comprises the sequence of SEQ ID NO:4, and the sense RNA strand comprises the sequence of SEQ ID NO:3. The cell may be a pancreatic carcinoma cell.
In another aspect, the invention relates to a method for inhibiting the expression of an anti-apoptotic gene in a cell. The method comprises introducing into the cell a dsRNA, as described above, then maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of the anti-apoptotic gene. The cell may be a pancreatic carcinoma cell.
In yet another aspect, the invention relates to a pharmaceutical composition for inhibiting the expression of an anti-apoptic gene in an organism. The pharmaceutical composition comprises a dsRNA, as described above, and a pharmaceutically acceptable carrier. The anti-apoptotic gene may be a Bcl gene, such Bcl-2, Bcl-w, or Bcl-xL. The cell may be a pancreatic carcinoma cell, and the organism may be a mammal, such as a human. The dosage unit of dsRNA in the pharmaceutical composition may be less than 5 milligram (mg) of dsRNA, preferably in a range of 0.01 to 2.5 milligrams (mg), more preferably 0.1 to 200 micrograms (μg), even more preferably 0.1 to 100 μg, and most preferably less than 25 μg per kilogram body weight of the mammal. The pharmaceutically acceptable carrier may be an aqueous solution, such phosphate buffered saline. The pharmaceutically acceptable carrier may comprise a micellar structure, such as a liposome, capsid, capsoid, polymeric nanocapsule, or polymeric microcapsule. In a preferred embodiment, the micellar structure is a liposome. The pharmaceutical composition may be formulated to be administered by inhalation, infusion, injection, or orally, preferably by intravenous or intraperitoneal injection.
In still another aspect, the invention relates to method for treating a disease caused by the expression of an anti-apoptotic gene in a mammal. The method comprises administering a pharmaceutical composition, as described above, to the mammal. The disease to be treated may be a pancreatic carcinoma.
The details of once or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the apoptosis rate (percent) of human pancreatic YAP C cancer cells, 120 hours after transfection with dsRNA 1 that is complementary to a first sequence of the human Bcl-2 gene.
FIG. 2 shows the apoptosis rate (percent) of YAP C cells, 120 hours after transfection with dsRNA 2 that is complementary to a first sequence of the human Bcl-2 gene.
FIG. 3 shows the apoptosis rate (percent) of YAP C cells, 120 hours after transfection with dsRNA 3 that is complementary to a sequence of the neomycin resistance gene.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of a target gene in a cell using the dsRNA. The present invention also discloses compositions and methods for treating diseases in organisms caused by the expression of an anti-apoptotic gene using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
The dsRNA of the invention comprises an RNA strand (the complementary strand) having a region which is less than 25 nucleotides in length and is complementary to at least a portion of an RNA transcript of an anti-apoptotic target gene, such as Bcl-2, Bcl-w, or Bcl-xL. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in uncontrolled cell or tissue growth. Using cell-based assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the target gene(s). Not only are lower dosages of dsRNA required as compared to traditional antisense RNA, but dsRNA affects apoptosis to such an extent that there is a noticeable reduction in both tumor size and number of tumor cells. Thus, the present invention encompasses these dsRNAs and compositions comprising dsRNA and their use for specifically silencing genes whose protein products either inhibit or prevent apoptosis in tumor cells. Moreover, the dsRNAs of the invention have no apparent effect on neighboring normal cells. Thus, the methods and compositions of the present invention comprising these dsRNAs are useful for treating cellular proliferative and/or differentiation disorders, such as cancer.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of target anti-apoptotic genes, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes. The pharmaceutical compositions of the present invention comprise a dsRNA having an RNA strand comprising a complementary region which is less than 25 nucleotides in length and is complementary to at least a portion of an RNA transcript of an anti-apoptotic target gene, together with a pharmaceutically acceptable carrier. The anti-apoptotic gene may be a member of the Bcl-2 family, such as Bcl-2, Bcl-w, or Bcl-xL. The pharmaceutical composition may comprise a combination of dsRNAs having regions complementary to a plurality of anti-apoptotic genes, for example a combination of Bcl-2, Bcl-xL, and/or Bcl-w. Since many types of tumor cells are known to express multiple anti-apoptotic genes, compositions comprising a combination of dsRNAs are particularly effective at inhibiting the development and/or growth of tumor cells.
Accordingly, certain aspects of the present invention relate to pharmaceutical compositions comprising the dsRNA of the present invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of a target anti-apoptotic gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of at least one of these anti-apoptotic genes.
I. Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below.
As used herein, “target gene” refers to a section of a DNA strand of a double-stranded DNA that is complementary to a section of a DNA strand, including all transcribed regions, that serves as a matrix for transcription. A target gene, usually the sense strand, is a gene whose expression is to be selectively inhibited or silenced through RNA interference. As used herein, the term “target gene” specifically encompasses any cellular gene or gene fragment whose expression or activity is associated with the inhibition or prevention of apoptosis. For example, the target gene may be a gene from the Bcl-2 gene family, such as Bcl-2, Bcl-w, and/or Bcl-xL.
The term “complementary RNA strand” (also referred to herein as the “antisense strand”) refers to the strand of a dsRNA which is complementary to an mRNA transcript that is formed during expression of the target gene, or its processing products. As used herein, the term “complementary nucleotide sequence” refers to the region on the complementary RNA strand that is complementary to an mRNA transcript of a portion of the target gene. “dsRNA” refers to a ribonucleic acid molecule having a duplex structure comprising two complementary and anti-parallel nucleic acid strands. Not all nucleotides of a dsRNA must exhibit Watson-Crick base pairs; the two RNA strands may be substantially complementary (i.e., having no more than one or two nucleotide mismatches). The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. The RNA strands may have the same or a different number of nucleotides. The dsRNA is less than 30, preferably less than 25, and most preferably between 21 and 24 nucleotides in length. dsRNAs of this length are particularly efficient in inhibiting the expression of the target anti-apoptotic gene. “Introducing into” means uptake or absorption in the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through cellular processes, or by auxiliary agents or devices. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro delivery includes methods known in the art such as electroporation and lipofection.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure when a 3′-end of one RNA strand extends beyond the 5′-end of the other strand, or vice versa.
As used herein and as known in the art, the term “identity” is the relationship between two or more polynucleotide sequences, as determined by comparing the sequences. Identity also means the degree of sequence relatedness between polynucleotide sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While there exist a number of methods to measure identity between two polynucleotide sequences, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York (1991)). Methods commonly employed to determine identity between sequences include, for example, those disclosed in Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48:1073. “Substantially identical,” as used herein, means there is a very high degree of homology (preferably 100% sequence identity) between the sense strand of the dsRNA and the corresponding part of the target gene. However, dsRNA having greater than 90%, or 95% sequence identity may be used in the present invention, and thus sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated. Although 100% identity is preferred, the dsRNA may contain single or multiple base-pair random mismatches between the RNA and the target gene.
As used herein, the term “treatment” refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a dsRNA molecule has been introduced by means of recombinant DNA techniques.
II. Double-stranded Ribonucleic Acid (dsRNA)
In one embodiment, the invention relates to a double-stranded ribonucleic acid (dsRNA) having a nucleotide sequence which is substantially identical to at least a portion of a target gene. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form the duplex structure. One strand of the dsRNA comprises the nucleotide sequence that is substantially identical to a portion of the target gene (the “sense” strand), and the other strand (the “complementary” or “antisense” strand) comprises a sequence that is complementary to an RNA tanscript of the target gene. The complementary region is less between 19 and 24, preferably between 21 and 23, and most preferably 22 nucleotides in length. The dsRNA is less than 30 nucleotides, preferably less than 25 nucleotides, and most preferably between 21 and 24 nucleotides in length. The dsRNA can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer, such as are commercially available from Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the target gene is a member of the Bcl-2 family, e.g., Bcl-2, Bcl-2, or Bcl-xL. In specific embodiments, the complementary (antisense) RNA strand of the dsRNA comprises the sequence set forth in SEQ ID NO:2 and the second (sense) RNA strand comprises the sequence set forth in SEQ ID NO:1; or the complementary (antisense) RNA strand of the dsRNA comprises the sequence set forth in SEQ ID NO:4 and the second (sense) RNA strand comprises the sequence set forth in SEQ ID NO:3.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without effecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Preferably, the single-stranded overhang is located at the 3′-terminal end of the complementary (antisense) RNA strand or, alternatively, at the 3′-terminal end of the second (sense) strand. The dsRNA may also have a blunt end, preferably located at the 5′-end of the complementary (antisense) strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Preferably, the complementary strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified for improved stability, i.e., enhanced resistance to degradation and/or strand dissociation. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. In one embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNAs are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a preferred embodiment, the 5′-end of the complementary (antisense) RNA strand and the 3′-end of the second (sense) RNA strand are chemically linked via a hexa-ethylene glycol linker.
III. Pharmaceutical Compositions Comprising dsRNA
In one embodiment, the invention relates to a pharmaceutical composition comprising a dsRNA, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of an anti-apoptotic gene.
In another embodiment, the invention relates to a pharmaceutical composition comprising at least two dsRNAs, designed to target different anti-apoptotic genes, and a pharmaceutically acceptable carrier. The anti-apoptotic genes may be members of the Bcl-2 family, such as Bcl-2, Bcl-w, or Bcl-xL. Due of the targeting of mRNA of multiple anti-apoptotic genes, pharmaceutical compositions comprising a plurality of dsRNAs may provide improved efficiency of treatment as compared to compositions comprising a single dsRNA, at least in tumor cells expressing these multiple genes. In this embodiment, the individual dsRNAs are prepared as described in the preceding section, which is incorporated by reference herein. One dsRNA may have a nucleotide sequence which is substantially identical to at least a portion of one anti-apoptotic gene; additional dsRNAs are prepared, each of which has a nucleotide sequence that is substantially identical to a portion of a different anti-apoptotic gene. For example, one dsRNA may have a nucleotide sequence that is substantially identical to a Bcl-2 gene, another dsRNA may have a nucleotide sequence that is substantially identical to a Bcl-xL gene, and yet another dsRNA may have a nucleotide sequence that is substantially identical to a Bcl-w gene. The multiple dsRNAs may be combined in the same pharmaceutical composition, or formulated separately. If formulated individually, the compositions containing the separate dsRNAs may comprise the same or different carriers, and may be administered using the same or different routes of administration. Moreover, the pharmaceutical compositions comprising the individual dsRNAs may be administered substantially simultaneously, sequentially, or at preset intervals throughout the day or treatment period. Although the foregoing description relates to target genes from the Bcl-2 family, the present invention encompasses any gene or combination of genes that have an inhibitory or preventive effect on apoptosis.
The pharmaceutical compositions of the present invention are administered in dosages sufficient to inhibit expression of the target gene. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of the target gene.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, more preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day, even more preferably in the range of 1.0 to 50 micrograms per kilogram body weight per day, and most preferably in the range of 1.0 to 25 micrograms per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases. For example, mouse models are available for hematopoictic malignancies such as leukemias, lymphomas and acute myclogenous leukemia. The MMHCC (Mouse models of Human Cancer Consortium) web page (emice.nci.nih.gov), sponsored by the National Cancer Institute, provides disease-site-specific compendium of known cancer models, and has links to the searchable Cancer Models Database (cancermodels.nci.nih.gov), as well as the NCI-MMHCC mouse repository. Examples of the genetic tools that are currently available for the modeling of leukemia and lymphomas in mice, and which are useful in practicing the present invention, are described in the following references: Maru, Y., Int. J. Hematol. (2001) 73:308-322; Pandolfi, P. P., Oncogene (2001) 20:5726-5735; Pollock, J. L., et al., Curr. Opin. Hematol. (2001) 8:206-211; Rego, E. M., et al., Semin. in Hemat. (2001) 38:4-70; Shannon, K. M., et al. (2001) Modeling myeloid leukemia tumors suppressor gene inactivation in the mouse, Semin. Cancer Biol. 11, 191-200; Van Etten, R. A., (2001) Curr. Opin. Hematol. 8, 224-230; Wong, S., et al. (2001) Oncogene 20, 5644-5659; Phillips J A., Cancer Res. (2000) 52(2):437-43; Harris, A. W., et al, J. Exp. Med. (1988) 167(2):353-71; Zeng X X et al., Blood. (1988) 92(10):3529-36; Eriksson, B., et al., Exp. Hematol. (1999) 27(4):682-8; and Kovalchuk, A. et al., J. Exp. Med. (2000) 192(8):1183-90. Mouse repositories can also be found at: The Jackson Laboratory, Charles River Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) National Network and at the European Mouse Mutant Archive. Such models may be used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.
For oral administration, the dsRNAs useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
For intramuscular, intraperitoneal, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of dsRNA in the cells that express the target gene. Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Surprisingly, the present inventors have discovered that compositions containing only naked dsRNA and a physiologically acceptable solvent are taken up by cells, where the dsRNA effectively inhibits expression of the target gene. Although microinjection, lipofection, viruses, viroids, capsids, capsoids, or other auxiliary agents are required to introduce dsRNA into cell cultures, surprisingly these methods and agents are not necessary for uptake of dsRNA in vivo. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the dsRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075, which are incorporated by reference herein.
In one embodiment, the encapsulated formulation comprises a viral coat protein. In this embodiment, the dsRNA may be bound to, associated with, or enclosed by at least one viral coat protein. The viral coat protein may be derived from or associated with a virus, such as a polyoma virus, or it may be partially or entirely artificial. For example, the coat protein may be a Virus Protein 1 and/or Virus Protein 2 of the polyoma virus, or a derivative thereof.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs useful according to the invention can be administered in combination with other known agents effective in treatment of diseases. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
For oral administration, the dsRNAs useful in the invention will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
IV. Methods for Treating Diseases Caused by Expression of an Anti-apoptotic Gene.
In one embodiment, the invention relates to a method for treating a subject having a disease or at risk of developing a disease caused by the expression of an anti-apoptotic target gene. In this embodiment, the dsRNA can act as novel therapeutic agents for controlling one or more of cellular proliferative and/or differentiative disorders. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of the target gene is silenced. Because of their high specificity, the dsRNAs of the present invention specifically target mRNAs of target genes of diseased cells and tissues, as described below, and at surprisingly low dosages.
In the prevention of disease, the target gene may be one which is required for initiation or maintenance of the disease, or which has been identified as being associated with a higher risk of contracting the disease. In the treatment of disease, the dsRNA can be brought into contact with the cells or tissue exhibiting the disease. For example, dsRNA substantially identical to all or part of a mutated gene associated with cancer, or one expressed at high levels in tumor cells, e.g. aurora kinase, may be brought into contact with or introduced into a cancerous cell or tumor gene.
Examples of cellular proliferative and/or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of pancreas, prostate, colon, lung, breast and liver origin. As used herein, the terms “cancer,” “hyperproliferative,” and “neoplastic” refer to cells having the capacity for autonomous growth, i.e., an abnormal state of condition characterized by rapidly proliferating cell growth. These terms are meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Proliferative disorders also include hematopoietic neoplastic disorders, including diseases involving hyperplastic/neoplatic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
Examples of genes which can be targeted for treatment include, without limitation, an oncogene (Hanahan, D. and R. A. Weinberg, Cell (2000) 100:57; and Yokota, J., Carcinogenesis (2000) 21(3):497-503); genes of proteins that are involved in metastasizing and/or invasive processes (Boyd, D., Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J., Carcinogenesis (2000) 21(3):497-503); genes of proteases as well as of molecules that regulate apoptosis and the cell cycle (Matrisian, L. M., Curr. Biol. (1999) 9(20):R776-8; Krepela, E., Neoplasma (2001) 48(5):332-49; Basbaum and Werb, Curr. Opin. Cell Biol. (1996) 8:731-738; Birkedal-Hansen, et al., Crit. Rev. Oral Biol. Med. (1993) 4:197-250; Mignatti and Rifkin, Physiol. Rev. (1993) 73:161-195; Stetler-Stevenson, et al., Annu. Rev. Cell Biol. (1993) 9:541-573; Brinkerhoff, E., and L. M. Matrisan, Nature Reviews (2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem. (2000) 69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol. (1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001) 488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996) 2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari, R., Bioassays (2001) 23(7):563-5); genes that express the EGF receptor; Mendelsohn, J. and J. Baselga, Oncogene (2000) 19(56):6550-65; Normanno, N., et al., Front. Biosci. (2001) 6:D685-707); and the multi-drug resistance 1 gene, MDR1 gene (Childs, S., and V. Ling, Imp. Adv. Oncol. (1994) 21-36).
In one embodiment, a pharmaceutical compositions comprising dsRNA is used to inhibit the expression of the multi-drug resistance 1 gene (“MDR1”). “Multi-drug resistance” (MDR) broadly refers to a pattern of resistance to a variety of chemotherapeutic drugs with unrelated chemical structures and different mechanisms of action. Although the etiology of MDR is multifactorial, the overexpression of P-glycoprotein (Pgp), a membrane protein that mediates the transport of MDR drugs, remains the most common alteration underlying MDR in laboratory models (Childs, S., Imp. Adv. Oncol. (1994) 21-36). Moreover, expression of Pgp has been linked to the development of MDR in human cancer, particularly in the leukemias, lymphomas, multiple myeloma, neuroblastoma, and soft tissue sarcoma (Fan., D., et al., Reversal of Multidrug Resistance in Cancer, ed. Kellen, J. A. (CRC, Boca Raton, Fla.), pp. 93-125). Recent studies showed that tumor cells expressing MDR-associated protein (MRP) (Cole, S. P. C., et al., Science (1992) 258:1650-1654) and lung resistance protein (LRP) (Scheffer, G. L., et al., Nat. Med. (1995)1:578-582) and mutation of DNA topoisomerase II (Beck, W. T., J. Natl. Cancer Inst. (1989) 81:1683-1685) also may render MDR.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection.
V. Methods for Inhibiting Expression of an Anti-apoptotic Gene
In yet another aspect, the invention relates to a method for inhibiting the expression of an anti-apoptotic gene in an organism. The method comprises administering a composition of the invention to the organism such that expression of the target anti-apoptotic gene is silenced. The organism may be an animal or a plant. Because of their high specificity, the dsRNAs of the present invention specifically target RNAs (primary or processed) of target anti-apoptotic genes, and at surprisingly low dosages. Compositions and methods for inhibiting the expression of these target genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the target anti-apoptotic gene of the organism to be treated. When the organism to be treated is a mammal, such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous or intraparenteral infusion or injection.
The methods for inhibiting the expression of a target gene can be applied to any gene or group of genes that have a direct or indirect inhibitory affect on apoptosis. Examples of human genes which can be targeted for silencing according to the methods of the present invention include, without limitation, an oncogene; a gene that expresses molecules that induce angiogenesis; genes of proteins that are involved in metastasizing and/or invasive processes; and genes of proteases as well as of molecules that regulate apoptosis and the cell cycle. In a preferred embodiment, the tumor disease to be treated is a pancreatic carcinoma. There is no known treatment for pancreatic cancer, which currently has a survival rate of approximately 3%, the lowest of all carcinomas.
The methods for inhibition the expression of a target gene can also be applied to any plant anti-apoptotic gene one wishes to silence, thereby specifically inhibiting its expression.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Inhibition of Bcl-2 Gene Expression by RNA Interference
The cells of the human pancreatic Yap C cancer line (German Microorganism and Cell Culture Collection, Braunschweig, (No. ACC 382)), were cultured at 37° C., 5% CO2 in RPMI 1640 medium (Biochrom Corp., Berlin) with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin. Human skin fibroblasts were cultured under the same conditions in Dulbecco's MEM with 10% FCS and 1% penicillin/streptomycin. The double-stranded oligoribonucleotides used for transfection have the following sequences, designated as SEQ ID NO:1 to SEQ ID No:6 in the sequence protocol:
dsRNA 1, which is complementary to a first sequence of the human Bcl-2 gene:
S2: 5′-cag gac cuc gcc gcu gca gac c-3′ (SEQ ID NO:1)
S1: 3′-cg guc cug gag egg cga cgu cug g-5′ (SEQ ID NO:2)
dsRNA 2, which is complementary to a second sequence of the human Bcl-2 gene:
S2: 5′-g ccu uug ugg aac ugu acg gcc-3′ (SEQ ID NO:3)
S1: 3′-uac gga aac acc uug aca ugc cgg-5′ (SEQ ID NO:4)
dsRNA 3, which is complementary to a sequence of the neomycin resistance gene:
S2: 5′-c aag gau gag gau cgu uuc gca-3′ (SEQ ID NO:5)
S1: 3′-ucu guc cua cuc cua gca aag cg-5′ (SEQ ID NO:6)
Transfection was carried out in a 6-well plate with oligofectamine (Invitrogen Corp., Karlsruhe). 250,000 cells were placed in each well. Transfection of the double-stranded oligoribonucleotides was carried out in accordance with the oligofectamine protocol recommended by Invitrogen (the data relate to 1 well of a 6-well plate): 10 μl of the double-stranded oligoribonucleotides (0.1-10 μM) were diluted with 175 μl cell culture medium without additives. 3 μl oligofectamine were diluted with 12 μl cell culture medium without additives, and incubated for 10 minutes at room temperature. The diluted oligofectamine was then added to the diluted double-stranded oligoribonucleotides, mixed, and incubated for 20 minutes at room temperature. During this time, the cells to be transfected were washed once with cell culture medium without additives, and 800 μl of fresh cell culture medium was added so that the transfection end volume was 1000 μl. This results in a double-stranded oligoribonucleotide end concentration of 1-100 μM. The transfection media was incubated with the cells for four hours at 37° C. 500 μl of cell culture medium with 30% FCS were then placed in each well, i.e. final concentration of FCS was 10%. The cells were then incubated for 120 hours at 37° C., at which time they were washed with phosphate buffered saline (PBS), trypsinized and centrifuged for 10 minutes at 100 g. The supernatant fluid was discarded, and the pellet was incubated in the dark with hypotonic propidium iodide solution for 30 minutes at 4° C. The pelletted cells were then analyzed by flow cytometry using a FACSCalibur fluorescence-activated cell sorter (BD GmbH, Heidelberg).
Both the double-stranded oligoribonucleotides dsRNA 1 and dsRNA 2 decreased the inhibition of apoptosis mediated by Bcl-2 in the human pancreatic cancer cells studied. No additional stimulation of apoptosis was required to induce or initiate apoptosis. The apoptosis rate rose independent of incubation time. FIG. 1 shows the result achieved with dsRNA 1 and FIG. 2 that with dsRNA 2. Whereas untreated YAP C control cells and cells with which the described methods of transfection were carried out without double-stranded oligoribonucleotides (mock-transfected cells) showed an apoptosis rate of only 3.8% and 7.1% after 120 hours incubation, the apoptosis rate achieved with 100 nM dsRNA rose to 37.2% for transfection with dsRNA 1 and 28.9% for transfection with dsRNA 2. Control transfection with dsRNA 3 led to a maximum apoptosis rate of 13.5%. This represents no significant increase when compared to mock-transfected cells, and proves the sequence specificity of the action of the dsRNA 1 and dsRNA 2. As a control, skin fibroblasts were transfected as non-transformed cells with dsRNA 1 and dsRNA 2. After 120 hours, these cells showed no significant increase in apoptosis rate.
Example 2
Treatment of a Pancreatic Cancer Patient with dsRNA 1 and 2
In this Example, dsRNA 1 and 2 are injected into a pancreatic cancer patient and shown to specifically inhibit Bcl-2 gene expression.
Synthesis and Preparation of dsRNAs
dsRNA 1 and 2 directed against the Bcl-2 gene are chemically synthesized with or without a hexaethylene glycol linker. Oligoribonucleotides are synthesized with an RNA synthesizer (Expedite 8909, Applied Biosystems, Weiterstadt, Germany) and purified by High Pressure Liquid Chromatography (HPLC) using NucleoPac PA-100 columns, 9×250 mm (Dionex Corp.; low salt buffer: 20 mM Tris, 10 mM NaClO4, pH 6.8, 10% acetonitrile; the high-salt buffer was: 20 mM Tris, 400 mM NaClO4, pH 6.8, 10% acetonitrile. flow rate: 3 ml/min). Formation of double stranded dsRNAs is then achieved by heating a stoichiometric mixture of the individual complementary strands (10 μM) in 10 mM sodium phosphate buffer, pH 6.8, 100 mM NaCl, to 80-90° C., with subsequent slow cooling to room temperature over 6 hours.
In addition, dsRNA molecules with linkers may be produced by solid phase synthesis and addition of hexaethylene glycol as a non-nucleotide linker (Jeremy, D., et al., Biochem. (1996), 35:14665-14670). A hexaethylene glycol linker phosphoramidite (Chruachem Ltd, Todd Campus, West of Scotland Science Park, Acre Road, Glasgow, G20 OUA, Scotland, UK) is coupled to the support bound oligoribonucleotide employing the same synthetic cycle as for standard nucleoside phosphoramidites (Proligo Biochemie GmbH, Georg-Hyken-Str.14, Hamburg, Germany) but with prolonged coupling times. Incorporation of linker phosphoramidite is comparable to the incorporation of nucleoside phosphoramidites.
dsRNA Administration and Dosage
The present example provides for pharmaceutical compositions for the treatment of human pancreatic cancer patients comprising a therapeutically effective amount of a dsRNA 1 and dsRNA 2 as disclosed herein, in combination with a pharmaceutically acceptable carrier or excipient. dsRNAs useful according to the invention may be formulated for oral or parenteral administration. The pharmaceutical compositions may be administered in any effective, convenient manner including, for instance, administration by topical, oral, anal, vaginal, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes among others. One of skill in the art can readily prepare dsRNAs for injection using such carriers that include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Additional examples of suitable carriers are found in standard pharmaceutical texts, e.g. “Remington's Pharmaceutical Sciences”, 16th edition, Mack Publishing Company, Easton, Pa. (1980).
RNA Purification and Analysis
Efficacy of the dsRNA treatment is determined at defined intervals after the initiation of treatment using real time PCR on total RNA extracted from tissue biopsies. Cytoplasmic RNA from tissue biopsies, taken prior to and during treatment, is purified with the help of the RNeasy Kit (Qiagen, Hilden) and Bcl-2 mRNA levels are quantitated by real time RT-PCR as described previously (Eder, M., et al., Leukemia (1999) 13:1383-1389; Scherr, M., et al., BioTechniques (2001) 31:520-526). Analysis of Bcl-2 mRNA levels before and during treatment by real time PCR, provides the attending physician with a rapid and accurate assessment of treatment efficacy as well as the opportunity to modify the treatment regimen in response to the patient's symptoms and disease progression.
Example 3
dsRNA Expression Vectors
In another aspect of the invention, Bcl-2 specific dsRNA molecules that interact with Bcl-2 target RNA molecules and modulate Bcl-2 gene expression activity are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be incorporated and inherited as a transgene integrated into the host genome. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strands of a dsRNA can be transcribed by promoters on two separate expression vectors and co-transfected into a target cell. Alternatively each individual strand of the dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In a preferred embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
The recombinant dsRNA expression vectors are preferably DNA plasmids or viral vectors. dsRNA expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus (for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129)); adenovirus (see, for example, Berkner, et al., BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or alphavirus as well as others known in the art. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. NatI. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. NatI. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573). Recombinant retroviral vectors capable of transducing and expressing genes inserted into the genome of a cell can be produced by transfecting the recombinant retroviral genome into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can be used to infect a wide variety of cells and tissues in susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and also have the advantage of not requiring mitotically active cells for infection.
The promoter driving dsRNA expression in either a DNA plasmid or viral vector of the invention may be a eukaryotic RNA polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g. CMV early promoter or actin promoter or U1 snRNA promoter) or preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7 promoter, provided the expression plasmid also encodes T7 RNA polymerase required for transcription from a T7 promoter. The promoter can also direct transgene expression to the pancreas (see, e.g. the insulin regulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
In addition, expression of the transgene can be precisely regulated, for example, by using an inducible regulatory sequence and expression systems such as a regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of transgene expression in cells or in mammals include regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D 1-thiogalactopyranoside (EPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the dsRNA transgene.
Preferably, recombinant vectors capable of expressing dsRNA molecules are delivered as described below, and persist in target cells. Alternatively, viral vectors can be used that provide for transient expression of dsRNA molecules. Such vectors can be repeatedly administered as necessary. Once expressed, the dsRNAs bind to target RNA and modulate its function or expression. Delivery of dsRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
DsRNA expression DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g. Oligofectamine) or non-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdowns targeting different regions of a single target gene or multiple target genes over a period of a week or more are also contemplated by the present invention. Successful introduction of the vectors of the invention into host cells can be monitored using various known methods. For example, transient transfection. can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection. of ex vivo cells can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
The dsRNA 1 and 2 molecules can also be inserted into vectors and used as gene therapy vectors for human patients. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
Example 4
Method of Determining an Effective Dose of a dsRNA
A therapeutically effective amount of a composition containing a sequence that encodes Bcl-2 specific dsRNA, (i.e., an effective dosage), is an amount that inhibits expression of the polypeptide encoded by the Bcl-2 target gene by at least 10 percent. Higher percentages of inhibition, e.g., 15, 20, 30, 40, 50, 75, 85, 90 percent or higher may be preferred in certain embodiments. Exemplary doses include milligram or microgram amounts of the molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram). The compositions can be administered one time per week for between about 1 to 10 weeks, e.g., between 2 to 8 weeks, or between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. In some cases transient expression of the dsRNA may be desired. When an inducible promoter is included in the construct encoding an dsRNA, expression is assayed upon delivery to the subject of an appropriate dose of the substance used to induce expression.
Appropriate doses of a composition depend upon the potency of the molecule (the sequence encoding the dsRNA) with respect to the expression or activity to be modulated. One or more of these molecules can be administered to an animal (e.g., a human) to modulate expression or activity of one or more target polypeptides. A physician may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
The efficacy of treatment can be monitored either by measuring the amount of the Bcl-2 target gene mRNA (e.g. using real time PCR) or the amount of polypeptide encoded by the target gene mRNA (Western blot analysis). In addition, the attending physician will monitor the symptoms associated with pancreatic cancer afflicting the patient and compare with those symptoms recorded prior to the initiation of dsRNA treatment.
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10384260
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alnylam europe ag
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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435/6
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Nov 24th, 2015 12:00AM
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Dec 9th, 2011 12:00AM
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https://www.uspto.gov?id=US09193973-20151124
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Compositions and methods for increasing erythropoietin (EPO) production
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The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting one or more EGLN genes, EGLN1, EGLN2 and/or EGLN3 and methods of using such dsRNA compositions to inhibit expression of these genes.
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9193973
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1. A composition comprising
(a) at least one double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, wherein said dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 26 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 26) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 27 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 27), and
(b) at least one double-stranded ribonucleic acid (dsRNA) for inhibiting expression EGLN3, wherein said dsRNA for inhibiting expression of EGLN3 comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 282 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 282) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 283 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 283).
2. A composition comprising
(a) at least one double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, wherein said dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 26 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 26) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 27 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 27), and
(b) at least one double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN2 comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 176 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 176) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 177 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 177).
3. The composition of claim 1 or claim 2, wherein each dsRNA targeting each of EGLN1, EGLN2 and EGLN3 comprises at least one modified nucleotide.
4. The composition of claim 3, wherein at least one of said modified nucleotides is chosen from the group consisting of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
5. The composition of claim 4, wherein said modified nucleotide is chosen from the group consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
6. The composition of claim 5, wherein the each of the sense:antisense strand pairs of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, EGLN2 and EGLN3 contains a region of complementarity between the sense and antisense strands which is at least 17 nucleotides in length.
7. The composition of claim 6, wherein the region of complementarity is between 19 and 21 nucleotides in length.
8. The composition of claim 7, wherein the region of complementarity is 19 nucleotides in length.
9. The composition of claim 1, wherein each strand of each of the sense:antisense strand pairs of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, EGLN2 and EGLN3 is no more than 30 nucleotides in length.
10. The composition of claim 9, wherein at least one strand of each of the sense:antisense strand pairs of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, EGLN2 and EGLN3 comprises a 3′ overhang of at least 1 nucleotide.
11. The composition of claim 10, wherein at least one strand of each of the sense:antisense strand pairs of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, EGLN2 and EGLN3 comprises a 3′ overhang of at least 2 nucleotides.
12. A composition comprising
(a) at least one double-strand ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, wherein said dsRNA comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 88 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 88) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 89 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 89), and
(b) at least one double-strand ribonucleic acid (dsRNA) for inhibiting expression of EGLN3, wherein said dsRNA for inhibiting expression of EGLN3, comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 346 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 346) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 347 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 347),
wherein each of the sense strand and antisense strand of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1 and EGLN3 is no more than 30 nucleotides in length and wherein at least one strand of each of the sense strand and antisense strand of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1 and EGLN3 comprises a 3′ overhang of at least 2 nucleotides.
13. A composition comprising
(a) at least one double-strand ribonucleic acid (dsRNA) for inhibiting expression of EGLN1 comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 88 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 88) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 89 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 89), and
(b) at least one double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN2 comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 240 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 240) and the antisense strand comprises at least 15 contiguous nucleotides of SEQ ID NO: 241 and differing by no more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 241), wherein each of the sense strand and antisense strand of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1 and EGLN2 is no more than 30 nucleotides in length and wherein at least one strand of each of the sense strand and antisense strand of each of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1 and EGLN2 comprises a 3′ overhang of at least 2 nucleotides.
14. The composition of claim 1 or claim 2, further comprising a ligand conjugated to the 3′ end of the sense strand of any of the double-stranded ribonucleic acid (dsRNA) for inhibiting expression of EGLN1, EGLN2 or EGLN3.
15. A pharmaceutical composition for inhibiting expression of an EGLN gene comprising the composition of claim 1.
16. The pharmaceutical composition of claim 15, further comprising a lipid formulation.
17. The pharmaceutical composition of claim 16, wherein the lipid formulation is a MC3 formulation.
18. A method of inhibiting EGLN1, EGLN2 and EGLN3 expression in a cell, the method comprising:
(a) introducing into the cell the composition of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an EGLN gene, thereby inhibiting expression of the EGLN gene in the cell.
19. The method of claim 18, wherein EGLN1, EGLN2 and EGLN3 expression are each inhibited by at least 30%.
20. A method of treating a disorder mediated by EGLN expression comprising administering to a human in need of such treatment a therapeutically effective amount of the composition of claim 1.
21. The method of claim 20, wherein the human has anemia or a condition associated with anemia.
22. The method of claim 21, wherein the anemia is selected from the group consisting of anemia due to B12 deficiency, anemia due to folate deficiency, anemia due to iron deficiency, hemolytic anemia, hemolytic anemia due to G-6-PD deficiency, idiopathic aplastic anemia, idiopathic autoimmune hemolytic anemia, immune hemolytic anemia, iegaloblastic anemia, pernicious anemia, secondary aplastic anemia, and sickle cell anemia.
23. The method of claim 21, wherein the condition associated with anemia is selected from the group consisting of pale skin, dizziness, fatigue, headaches, irritability, low body temperature, numb/cold hands or feet, rapid heartbeat, reduced erythropoietin, shortness of breath, weakness and chest pain.
24. The method of claim 20, wherein the human has a disorder selected from the group consisting of hypoxia, a neurological condition, renal disease or failure, and cancers of the blood, bone and marrow.
25. A method of increasing erythropoietin levels in a cell or organism comprising contacting said cell or organism with the composition of claim 1.
26. A method of increasing erythropoietin levels in a cell or organism comprising contacting said cell or organism with the composition of claim 2.
27. A method of increasing erythropoietin levels in a cell or organism comprising contacting said cell or organism with the composition of claim 12 or 13.
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CROSS REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. §371 U.S. National Stage Entry of International Application No. PCT/US2011/064121 filed Dec. 9, 2011, which claims the benefit of priority of U.S. Provisional Application No. 61/421,727 filed Dec. 10, 2010, and U.S. Provisional Application No. 61/493,651 filed Jun. 6, 2011, the contents of which are each incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made, in part, with government support under Contract Number NIH CA068490 awarded by the National Institutes of Health. The government has certain rights in the invention.
REFERENCE TO SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a filed entitled 20021004US371SEQLST.txt created on Dec. 8, 2011 which is 632,864 bytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The invention relates to the specific inhibition of the expression of EGLN genes.
BACKGROUND OF THE INVENTION
Erythropoietin (EPO) is a hormone found in the plasma which regulates red cell production by promoting erythroid differentiation and initiating hemoglobin synthesis. The gene is in the EPO/TPO family and encodes a secreted, acidic glycosylated cytokine.
Recombinant human erythropoietin (EPO) has been used since 1986 to treat the anemia of chronic and end-stage kidney disease (Eschbach, et al., N. Engl. J. Med. 1987 Jan. 8; 316(2):73-8). However, this treatment is costly and requires parenteral administration. It has recently been linked to cardiovascular side effects (J. Bohlius et al., Lancet 373, 1532 (2009) and antibodies which form against EPO can result in Pure Red Cell Aplasia (PRCA), an uncommon condition which develops in association with a failure of the bone marrow to manufacture red blood cells, leaving patients with severe, treatment-resistant anemia (reported by Casadevall, et al, New England Journal of Medicine, Feb. 14, 2002).
In addition to its role as a kidney cytokine regulating hematopoiesis, EPO is also produced in the brain after oxidative or nitrosative stress. The transcription factor HIF1 (hypoxia inducible factor 1) is known to upregulate EPO following hypoxic stimuli (Digicaylioglu, M., Lipton, S. A. Nature 412: 641-647, 2001). This upregulation provides protection against apoptosis of erythroid progenitors in bone marrow and also apoptosis of brain neurons (Siren, A.-L., et al., Proc. Nat. Acad. Sci. 98: 4044-4049, 2001). Grimm et al. showed in the adult mouse retina that acute hypoxia dose-dependently stimulates expression of EPO, fibroblast growth factor-2, and vascular endothelial growth factor via HIF1 stabilization (Nature Med. 8: 718-724, 2002).
Further controlling the regulation of EPO production are a family of prolyl hydroxylases, the PHD proteins, which act to regulate the HIF transcription factors. PHD (prolyl hydroxylases) proteins belong to a superfamily of several 2-oxoglutarate-dependent dioxygenases (Kaelin Jr., and Ratcliffe, Mol. Cell. 30, 393 (2008). In the mouse, these genes are known as EGLN1 (PHD2, prolyl hydroxylase domain-containing protein 2 and by the synonyms hif-prolyl hydroxylase 2; hifph2; hph2; chromosome 1 open reading frame 12; c1orf12; sm20, rat, homolog of sm20; zinc finger mynd domain-containing protein 6; and zmynd6), EGLN2 (PHD1, prolyl hydroxylase domain-containing protein 1; and by the synonyms hif-prolyl hydroxylase 1; hifph1) and EGLN3 (PHD3 prolyl hydroxylase domain-containing protein 3; and by the synonyms hif-prolyl hydroxylase 3; hifph3). In an attempt to elucidate the function of PHD enzymes in hepatic EPO production, Minamishima et al., created knockout mice lacking liver expression of PHD1, PHD2, PHD3, or combinations thereof (Mol. Cell. Biol. 29, 5729 (2009)).
Subsequent studies by Minamishima and Kaelin using the knock-out model, suggested that while hepatic inactivation of PHD1, PHD2, or PHD3 alone did not increase EPO or hematocrit values, loss of all three PHDs increased both measurements (Science, 329, 407 and Supplemental Information (2010)). According to Minamishima, questions remain regarding the promoters used and the role that PHD2 plays (and at which developmental stage) independent of the other two enzymes in the activation of EPO production.
Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant or unwanted regulation of a gene.
Given the drawbacks of complete gene knockout and the inherent problems translating gene knockout to human therapy, the present invention contemplates the use of RNAi to effect gene modulation with improved outcomes in the production of erythropoietin.
During development the liver is the major source of EPO but over time eventually the liver EPO is switched off and in normal healthy adults their kidney makes the EPO to support normal red blood cell production. However, two to four million Americans with renal disease suffer from anemia due to impaired EPO production. If it is possible to turn on hepatic EPO using siRNA targeting EGLN genes the liver could now supply the EPO required to support red blood cell production to compensate for the damaged kidney function. Furthermore, using siRNA in LNPs it may be possible to activate fetally expressed genes in liver by targeting negative regulators of the pathway. This strategy could be used in the treatment of many other diseases and not just exclusively anemia.
SUMMARY OF THE INVENTION
Described herein are compositions and methods that effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of one or more of the EGLN genes, such as in a cell or mammal Also described are compositions and methods for treating pathological conditions and diseases caused by or associated with the expression of said genes, such as anemia, hypoxia, neurological conditions including degeneration, renal disease or failure, and cancers including those of the blood, bone and marrow. It has been discovered that synergistic effects are seen upon the administration of a mix or plurality of iRNA agents collectively targeting all three EGLN genes.
As used herein, the term “iRNA” refers to one or more agents that contain RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of expression of at least one EGLN gene in a cell or mammal. Alternatively, in another embodiment, an iRNA as described herein activates EGLN expression in a cell or mammal. It should be understood that as used herein the term “EGLN” refers to any of the EGLN genes in any mammalian species and having any of the synonyms referred to in the art. Where a specific species or gene variant is being referred to, the variant will be called out by name.
The iRNAs included in the compositions featured herein encompass a dsRNA having an RNA strand (the antisense strand) having a region that is 30 nucleotides or less, generally 19-24 nucleotides in length, that is substantially complementary to at least part of an mRNA transcript of an EGLN gene.
In one embodiment, an iRNA for inhibiting expression of an EGLN gene includes at least two sequences that are complementary to each other. The iRNA includes a sense strand having a first sequence and an antisense strand having a second sequence. The antisense strand includes a nucleotide sequence that is substantially complementary to at least part of an mRNA encoding EGLN, and the region of complementarity is 30 nucleotides or less, and at least 15 nucleotides in length. Generally, the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length. In some embodiments the iRNA is from about 15 to about 25 nucleotides in length, and in other embodiments the iRNA is from about 25 to about 30 nucleotides in length. The iRNA, upon contacting with a cell expressing EGLN, inhibits the expression of an EGLN gene by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more, such as when assayed by a method as described herein. In one embodiment, where contacting is by a mix or plurality of EGLN iRNAs, the expression of each EGLN gene is inhibited by at least 10%, at least 20%, at least 25%, at least 30%, at least 35% or at least 40% or more and inhibition need not be the same for each EGLN targeted by the mix. For example, a mix of iRNAs targeting EGLN1, 2 and 3 may result in inhibition of expression of EGLN1 by 10%, EGLN2 by 20% and EGLN3 by 10%. As such, the mix inhibits EGLN expression by at least 10%. In one embodiment, the EGLN iRNA or iRNAs are formulated in a stable nucleic acid lipid particle (SNALP).
The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a histogram showing the in vitro screening results of the EGLN 1, 2, and 3 genes. AD (duplex) numbers are those listed in Tables 2A-F. The additional digit listed in the figure after the decimal (“.”) point is an internal tracking number and may be disregarded when making reference to the duplexes listed in the tables.
FIG. 2 is a histogram showing the in vitro dose response screening results of the EGLN 1, 2, and 3 genes. AD (duplex) numbers are those listed in Tables 2A-F. The additional digit listed in the figure after the decimal (“.”) point is an internal tracking number and may be disregarded when making reference to the duplexes listed in the tables.
FIG. 3 is a histogram showing the specificity of knockdown of EGLN genes by the iRNA agents of the invention. Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894 for EGLN1 and the effect of the 3-iRNA mix. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773 for EGLN2 and the effect of the 3-iRNA mix. Panel 3 shows the specificity of the EGLN3 iRNA agent, AD-40758 for EGLN3 and the effect of the 3-iRNA mix.
FIG. 4 shows results from an ELISA assay. FIG. 4A shows a histogram of EPO production in pg/mL Erythropoietin production upon treatment with EGLN dsRNA. FIG. 4B shows a histogram of the ELISA results of treatment groups PBS (1-4 and average), Luciferase control (AD1955) (1-5 and average) and the 3-iRNA mix of EGLN 1, 2 and 3 targeting agents, AD-40894, AD-40773 and AD40758, respectively (1-5 and average). Each bar (except for the averages) represents an individual animal.
FIG. 5 is a histogram showing the specificity of knockdown of EGLN genes by the iRNA agents of the invention in a dose response study (mg per kg). Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894 for EGLN1. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773 for EGLN2. Panel 3 shows the specificity of the EGLN3 iRNA agent, AD-40758 for EGLN3. Each panel also shows the knockdown of the respective EGLN gene using a dual iRNA agent mix (AD-04894 and AD-40758, “94/58” in amounts of 67% and 33% “0.67/0.33”)
FIG. 6 is a histogram of the Week 1 hematology results showing reticuloctye and RBC levels upon treatment with a composition comprising an EGLN1-3 mix of iRNA agents.
FIG. 7 is a histogram of the Week 1 hematology results showing hemogolobin and hematocrit levels upon treatment with a composition comprising an EGLN1-3 mix of iRNA agents.
FIG. 8 is a histogram of the Week 2 hematology results showing reticuloctye and RBC levels upon treatment with a composition comprising an EGLN1-3 mix of iRNA agents.
FIG. 9 is a histogram of the Week 2 hematology results showing hemogolobin and hematocrit levels upon treatment with a composition comprising an EGLN1-3 mix of iRNA agents.
FIG. 10 is a histogram showing the increase of EPO mRNA after 2 doses at day 10.
FIG. 11 is a histogram showing the specificity of knockdown of EGLN genes by the iRNA agents of the invention in a dose response study (mg per kg). Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894 for EGLN1. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773 for EGLN2. Panel 3 shows the specificity of the EGLN3 iRNA agent, AD-40758 for EGLN3. Each panel also shows the knockdown of the respective EGLN gene using single iRNA agent mixes (AD-40894 is “EGLN1,” AD-40773 is “EGLN2” and AD-40758 is “EGLN3”), dual iRNA agent mixes (AD-40894 and AD-40773 is “EGLN 1+2,” AD-04894 and AD-40758 is “EGLN 1+3,” AD-40773 and AD-40758 is “EGLN 2+3”) and a trip iRNA agent mix (AD-40894, AD-40773 and AD-40758 is “EGLN 1+2+3”).
FIG. 12 is a histogram showing the effects on erythropoietin production by the iRNA agents of the invention in a dose response study (mg per kg). Panel 1 shows a histogram of the ELISA results of treatment groups PBS, Luciferase control (AD1955), single iRNA agent mixes, dual iRNA agent mixes and triple iRNA mixture. Panel 2 shows the increase of EPO mRNA in the iRNA mixtures which contain the EGLN1 iRNA agent (AD-40773) from the treatment groups PBS, Luciferase control (AD1955), single iRNA agent mixes, dual iRNA agent mixes and triple iRNA mixture. It is to be noted that E1 means the same as EGLN1, E2 means the same as EGLN2 and E3 means EGLN3.
FIG. 13 is a histogram of the hematology results showing hemogolobin, hematocrit, reticulocyte and red blood cell levels upon a two dose treatment with a composition of single iRNA agents, dual iRNA agents or a triple iRNA agent mixture, a luciferase control iRNA agent and PBS control.
FIG. 14 is a histogram of the regulation of hepcidin upon a two dose treatment with a composition of single iRNA agents, dual iRNA agents or a triple iRNA agent mixture, a luciferase control iRNA agent and PBS control.
FIG. 15 is a histogram showing tissue specificity in a dose response study (mg per kg). Panel 1 shows a histogram of the results of treatment groups Luciferase control (AD1955 is “LUC”), and a triple iRNA mixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) on EGLN1 found in the liver, kidney and spleen. Panel 2 shows a histogram of the results of treatment groups Luciferase control (AD1955 is “LUC”), and a triple iRNA mixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) on EGLN2 found in the liver, kidney and spleen. Panel 3 shows a histogram of the results of treatment groups Luciferase control (AD1955 is “LUC”), and a triple iRNA mixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) on EGLN3 found in the liver, kidney and spleen. Panel 4 shows an increase of EPO mRNA in the liver from the triple iRNA mixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) as compared to the Luciferase control (AD1955 is “LUC”) which was not seen in the kidney or spleen. The y-axis represents ratio of EPO to GAPDH mRNA levels in arbitrary units.
FIG. 16 is a line graph showing the durable effects of a cocktail (AD-40894 at 0.375 mg/kg, AD-40773 at 0.75 mg/kg and AD-40758 at 0.375 mg/kg) in a single dose injection or a double dose injection as compared to a Luciferase control (AD1955). Panel 1 shows the levels of EPO found after a single or double injection as compared to the control (LUC). Panel 2 shows that the injection of the cocktail can increase the amount hematocrit in the mouse for about a month after a single injection.
FIG. 17 is a histogram showing knockdown of EGLN genes by the iRNA agents of the invention. Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894 for EGLN1 (AD-40894), EGLN1-2 (mix of AD-40894 and AD-40773) and the effect of the 3-iRNA mix. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773 for EGLN1 (AD-40894), EGLN1-2 (mix of AD-40894 and AD-40773) and the effect of the 3-iRNA mix. Panel 3 shows the specificity of the EGLN3 iRNA agent, AD-40758 for EGLN1 (AD-40894), EGLN1-2 (mix of AD-40894 and AD-40773) and the effect of the 3-iRNA mix.
FIG. 18 is a histogram a summary of the downregulation of hepcidin by the iRNA agents of the invention.
FIG. 19 is a histogram showing the increase of EPO mRNA after 3 doses at day 12 in the animals who received the EGLN1-2-3 (mix of AD-40894, AD-40773 and AD-40758).
FIG. 20 is a scatter chart of the hematocrit levels for pre- and post-dose of the iRNA agents of the invention. Panel 1 is the baseline hematocrit levels of the animals at day 0. Panel 2 is the hematocrit levels of the animals on day 12.
FIG. 21 is a histogram of the hematology results showing hemogolobin, hematocrit, reticulocyte and red blood cell levels upon a three dose treatment with a composition of a single iRNA agent (EGLN1), dual iRNA agent (EGLN1+2) or a triple iRNA agent mixture (EGLN1+2+3), a luciferase control iRNA agent, a PBS control and a SHAM control.
FIG. 22 is a scatter chart of the iron parameters of animals upon a three dose treatment with a composition of a single iRNA agent (EGLN1), dual iRNA agent (EGLN1−2) or a triple iRNA agent mixture (EGLN1−2−3), a luciferase control iRNA agent, a PBS control and a SHAM control. Panel 1 shows the serum levels of iron in the animals. Panel 2 shows the transferrin saturation (TSAT), which is the ratio of serum iron and total iron-binding capacity multiplied by 100, of the individual animals. Panel 3 is the unsaturated iron binding capacity (UIBC) of the animals. Panel 4 is the total iron binding capacity (TIBC) of the animals. Panel 5 shows the ferritin level of the animals.
FIG. 23 shows the targeting of EglN genes rescues anemia caused by renal failure. (A) Overview of 5/6 nephrectomy procedure and dosing schedule. (B and C) Hemoglobin (B) and Hematocrit (C) levels in mice treated as depicted in (A).
FIG. 24 shows histograms of the hematologic data showing EPO and HAMP1 mRNA values at day 12 in mice treated with the indicated siRNAs as depicted in (A). HAMP1=hepcidin antimicrobrial peptide 1. mRNA levels were normalized to actin mRNA and then to corresponding sham mRNA level.
FIG. 25 is a histogram showing the reduction of anemia in rats. Panel A shows an effective knockdown of EGLN1 using the EGLN1/2 siRNAs of the present invention. Panel B shows an effective knockdown of EGLN2 using the EGLN1/2 siRNAs of the present invention. Panel C shows a decrease in hepcidin (HAMP1) levels in rats treated with the EGLN1/2 siRNAs of the present invention.
FIG. 26 shows bioluminescent images of HIF 1 alpha-Luc mice 72 hours after a single intravenous dose of LNPs targeting all three EglN family members or, as a negative control, green fluorescent protein (GFP). Total dose=1 mg/kg (0.33 mg/kg per family member).
DETAILED DESCRIPTION
Described herein are iRNAs and methods of using them for inhibiting the expression of one or more EGLN genes in a cell or a mammal where the iRNA targets the one or more EGLN genes. Also described are compositions and methods for treating pathological conditions and diseases caused by or associated with the expression of said genes, such as anemia, hypoxia, neurological conditions including degeneration, renal disease or failure, and cancers including those of the blood, bone and marrow. It has surprisingly been discovered that synergistic effects are seen upon the administration of a mix or plurality of iRNA agents collectively targeting all three EGLN genes.
The iRNAs of the compositions featured herein include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an EGLN gene. The use of these iRNAs enables the targeted degradation of mRNAs of genes that are implicated in pathologies associated with EGLN expression in mammals and with the signaling pathways involved in production of erythropoietin. Very low dosages of EGLN iRNAs in particular can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of one or more EGLN genes. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting EGLN can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of an EGLN gene. More surprising is the discovery by the present inventors of a mix or cocktail of iRNA agents which can specifically target EGLN 1, 2 and 3 and which can increase or stimulate erythropoietin production in a cell or organism. Thus, methods and compositions including these iRNAs are useful for treating pathological processes that can be mediated by down regulating EGLN genes or those which are associated with low EPO levels. The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of one or more EGLN genes, as well as compositions and methods for treating diseases and disorders caused by or modulated by the expression of this gene. Embodiments of the pharmaceutical compositions featured in the invention include an iRNA having an antisense strand comprising a region which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part of an RNA transcript of an EGLN gene, together with a pharmaceutically acceptable carrier. Embodiments of compositions featured in the invention also include an iRNA having an antisense strand having a region of complementarity which is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and is substantially complementary to at least part of an RNA transcript of an EGLN gene.
Accordingly, in some aspects, pharmaceutical compositions containing one or more EGLN iRNA agents and a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of an EGLN gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of an EGLN gene are featured in the invention.
I. Definitions
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.
As used herein, “EGLN” (“EGL Nine Homolog”) refers to any one or all of the group of EGLN genes. In the mouse, these genes are known as EGLN1 (PHD2, prolyl hydroxylase domain-containing protein 2 and by the synonyms hif-prolyl hydroxylase 2; hifph2; hph2; chromosome 1 open reading frame 12; c1orf12; sm20, rat, homolog of; sm20; zinc finger mynd domain-containing protein 6; and zmynd6), EGLN2 (PHD1, prolyl hydroxylase domain-containing protein 1; and by the synonyms hif-prolyl hydroxylase 1; hifph1) and EGLN3 (PHD3 prolyl hydroxylase domain-containing protein 3; and by the synonyms hif-prolyl hydroxylase 3; hifph3). The sequences of the mouse EGLN mRNA transcripts can be found at NM—053207.2 (EGLN1; SEQ ID NO: 5), NM—053208.4 (EGLN2; SEQ ID NO: 6) and NM—028133.2 (EGLN3; SEQ ID NO: 7). The sequence of a human EGLN mRNA transcripts can be found at NM—022051.2 (EGLN1); NM—053046.2 (EGLN2) and NM—022073.3 (EGLN3).
As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of EGLN expression. Alternatively, in another embodiment, an iRNA as described herein activates EGLN expression.
As used herein, the term “iRNA mix” or “iRNA cocktail” refers to a composition that comprises more than one iRNA. The iRNA mixes or cocktails of the present invention may comprise one or more iRNA agents to a single EGLN gene or may comprise one or more iRNA agents targeted to more than one EGLN gene. Where an iRNA mix or cocktail contains only iRNA agents targeting one or more EGLN genes, this mix may be referred to as an “EGLN mix” or “EGLN cocktail.”
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an EGLN gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion. For example, the target sequence will generally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides in length, including all sub-ranges therebetween. As non-limiting examples, the target sequence can be from 15-30 nucleotides, 15-26 nucleotides, 15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20 nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides, 18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22 nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides, 19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21 nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides, 20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22 nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides, 21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22 nucleotides.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes described herein.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs includes, but are not limited to, G:U Wobble or Hoogstein base pairing.
The terms “complementary,” “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding an EGLN protein). For example, a polynucleotide is complementary to at least a part of an EGLN mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding EGLN.
The term “double-stranded RNA” or “dsRNA,” as used herein, refers to an iRNA that includes an RNA molecule or complex of molecules having a hybridized duplex region that comprises two anti-parallel and substantially complementary nucleic acid strands, which will be referred to as having “sense” and “antisense” orientations with respect to a target RNA. The duplex region can be of any length that permits specific degradation of a desired target RNA through a RISC pathway, but will typically range from 9 to 36 base pairs in length, e.g., 15-30 base pairs in length. Considering a duplex between 9 and 36 base pairs, the duplex can be any length in this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 and any sub-range therein between, including, but not limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs, 15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs, 15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs, 18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs, 19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs, 19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs, 20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs, 20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs, 21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAs generated in the cell by processing with Dicer and similar enzymes are generally in the range of 19-22 base pairs in length. One strand of the duplex region of a dsDNA comprises a sequence that is substantially complementary to a region of a target RNA. The two strands forming the duplex structure can be from a single RNA molecule having at least one self-complementary region, or can be formed from two or more separate RNA molecules. Where the duplex region is formed from two strands of a single molecule, the molecule can have a duplex region separated by a single stranded chain of nucleotides (herein referred to as a “hairpin loop”) between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure. The hairpin loop can comprise at least one unpaired nucleotide; in some embodiments the hairpin loop can comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides. Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than a hairpin loop, the connecting structure is referred to as a “linker.” The term “siRNA” is also used herein to refer to a dsRNA as described above.
The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure or in the ribose-phosphate backbone structure, e.g., as described herein below. However, the molecules comprising ribonucleoside analogs or derivatives must retain the ability to form a duplex. As non-limiting examples, an RNA molecule can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleostide, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoro modified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modified nucleoside, morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, an RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more, up to the entire length of the dsRNA molecule. The modifications need not be the same for each of such a plurality of modified ribonucleosides in an RNA molecule. In one embodiment, modified RNAs contemplated for use in methods and compositions described herein are peptide nucleic acids (PNAs) that have the ability to form the required duplex structure and that permit or mediate the specific degradation of a target RNA via a RISC pathway.
In one aspect, a modified ribonucleoside includes a deoxyribonucleoside. In such an instance, an iRNA agent can comprise one or more deoxynucleosides, including, for example, a deoxynucleoside overhang(s), or one or more deoxynucleosides within the double stranded portion of a dsRNA. However, it is self evident that under no circumstances is a double stranded DNA molecule encompassed by the term “iRNA.”
In one aspect, an RNA interference agent includes a single stranded RNA that interacts with a target RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into plants and invertebrate cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleaves the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA that promotes the formation of a RISC complex to effect silencing of the target gene.
As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) may be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′ end, 3′ end or both ends of either an antisense or sense strand of a dsRNA.
In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/or the 5′ end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.
The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches may be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.
As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP represents a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817. These applications are incorporated herein by reference in their entirety.
“Introducing into a cell,” when referring to an iRNA, means facilitating or effecting uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; an iRNA may also be “introduced into a cell,” wherein the cell is part of a living organism. In such an instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or known in the art.
As used herein, the term “modulate the expression of,” refers to at an least partial “inhibition” or partial “activation” of one or more EGLN gene expression in a cell treated with an iRNA composition as described herein compared to the expression of the one or more EGLN genes in an untreated cell.
The terms “activate,” “enhance,” “up-regulate the expression of,” “increase the expression of,” and the like, in so far as they refer to an EGLN gene, herein refer to the at least partial activation of the expression of an EGLN gene, as manifested by an increase in the amount of EGLN mRNA, which may be isolated from or detected in a first cell or group of cells in which an EGLN gene is transcribed and which has or have been treated such that the expression of an EGLN gene is increased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells).
In one embodiment, expression of an EGLN gene is activated by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA as described herein. In some embodiments, an EGLN gene is activated by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, expression of an EGLN gene is activated by at least about 85%, 90%, or 95% or more by administration of an iRNA as described herein. In some embodiments, EGLN gene expression is increased by at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000 fold or more in cells treated with an iRNA as described herein compared to the expression in an untreated cell. Activation of expression by small dsRNAs is described, for example, in Li et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and in US20070111963 and US2005226848, each of which is incorporated herein by reference.
The terms “silence,” “inhibit the expression of,” “down-regulate the expression of,” “suppress the expression of,” and the like, in so far as they refer to an EGLN gene, herein refer to the at least partial suppression of the expression of an EGLN gene, as manifested by a reduction of the amount of EGLN mRNA which may be isolated from or detected in a first cell or group of cells in which an EGLN gene is transcribed and which has or have been treated such that the expression of an EGLN gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of
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Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to EGLN gene expression, e.g., the amount of protein encoded by an EGLN gene, or the number of cells displaying a certain phenotype, e.g., lack of or decreased cytokine production. In principle, EGLN gene silencing may be determined in any cell expressing EGLN, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given iRNA inhibits the expression of an EGLN gene by a certain degree and therefore is encompassed by the instant invention, the assays provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of an EGLN gene is suppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administration of an iRNA featured in the invention. In some embodiments, an EGLN gene is suppressed by at least about 60%, 70%, or 80% by administration of an iRNA featured in the invention. In some embodiments, an EGLN gene is suppressed by at least about 85%, 90%, 95%, 98%, 99%, or more by administration of an iRNA as described herein.
As used herein in the context of EGLN expression, the terms “treat,” “treatment,” and the like, refer to relief from or alleviation of pathological processes mediated by EGLN expression. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by EGLN expression), the terms “treat,” “treatment,” and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression or anticipated progression of such condition, such as slowing the progression of a malignancy or cancer, treating anemia, hypoxia, neurological conditions including degeneration, renal disease or failure, and cancers including those of the blood, bone and marrow.
By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of pathological processes mediated by EGLN expression or an overt symptom of pathological processes mediated by EGLN expression. In one embodiment, a therapeutically effective amount is that amount of iRNA agent or agents which result in the increased production of erythropoietin in the system being treated. The specific amount that is therapeutically effective can be readily determined by an ordinary medical practitioner, and may vary depending on factors known in the art, such as, for example, the type of pathological processes mediated by EGLN expression, the patient's history and age, the stage of pathological processes mediated by EGLN expression, and the administration of other agents that inhibit pathological processes mediated by EGLN expression.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of an iRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an iRNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 10% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 10% reduction in that parameter. For example, a therapeutically effective amount of an iRNA targeting EGLN can reduce EGLN protein levels by at least 10% or may result in the increase in EPO production by at least 1%, 5%, 10% or more.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract. Agents included in drug formulations are described further herein below.
II. Double-stranded Ribonucleic Acid (dsRNA)
Described herein are iRNA agents that inhibit the expression of one or more EGLN genes. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an EGLN gene in a cell or mammal, e.g., in a human having anemia, hypoxia, neurological conditions including degeneration, renal disease or failure, or cancers including those of the blood, bone and marrow where the dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an EGLN gene, and where the region of complementarity is 30 nucleotides or less in length, generally 19-24 nucleotides in length, and where the dsRNA, upon contact with a cell expressing the EGLN gene, inhibits the expression of the EGLN gene by at least 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by Western blot. In one embodiment, the iRNA agent activates the expression of an EGLN gene in a cell or mammal Expression of an EGLN gene in cell culture, such as in COS cells, HeLa cells, primary hepatocytes, kidney cells, HEK-293 cells, MDCK cells, HepG2 cells, primary cultured cells or in a biological sample from a subject can be assayed by measuring EGLN mRNA levels, such as by bDNA or TaqMan assay, or by measuring protein levels, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.
A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of an EGLN gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of 9 to 36, e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex of e.g., 15-30 base pairs that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, then, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target EGLN expression is not generated in the target cell by cleavage of a larger dsRNA.
A dsRNA as described herein may further include one or more single-stranded nucleotide overhangs. The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In one embodiment, an EGLN gene is a human EGLN gene. In another embodiment the EGLN gene is a mouse or a rat EGLN gene. In specific embodiments, the first sequence is a sense strand of a dsRNA that includes a sense sequence from Tables 2A-F and 6A-C, and the second sequence is selected from the group consisting of the corresponding antisense sequences of Tables 2A-F and 6A-C. Alternative dsRNA agents that target elsewhere in the target sequence provided in Tables 2A-F and 6A-C can readily be determined using the target sequence and the flanking EGLN sequence.
In one aspect, a dsRNA will include at least nucleotide sequences, whereby the sense strand is selected from the groups of sequences provided in Tables 2A-F and 6A-C, and the corresponding antisense strand of the sense strand selected from Tables 2A-F and 6A-C. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an EGLN gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Tables 2A-F and 6A-C, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand from Tables 2A-F and 6A-C. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.
The skilled person is well aware that dsRNAs having a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2A-F and 6A-C, dsRNAs described herein can include at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2A-F and 6A-C minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 2A-F and 6A-C, and differing in their ability to inhibit the expression of an EGLN gene by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated according to the invention.
In addition, the RNAs provided in Tables 2A-F and 6A-C identify a site in an EGLN transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of such sequences. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least 15 contiguous nucleotides from one of the sequences provided in Tables 2A-F and 6A-C coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an EGLN gene.
While a target sequence is generally 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that may serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2A-F and 6A-C represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.
Further, it is contemplated that for any sequence identified, e.g., in Tables 2A-F and 6A-C, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those and sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) as an expression inhibitor.
An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide iRNA agent RNA strand which is complementary to a region of an EGLN gene, the RNA strand generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an EGLN gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an EGLN gene is important, especially if the particular region of complementarity in an EGLN gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of a dsRNA has a single-stranded nucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in this invention include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.
Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.
Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No. RE39,464, each of which is herein incorporated by reference
Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.
In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.
Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2—[known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2—[wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified RNAs may also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include O[(CH2)nO]mCH3, O(CH2).nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Hely. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2, also described in examples herein below.
Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.
An iRNA may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.
Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 513,030; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.
The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.
Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.
Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.
The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.
In one ligand, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).
In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:1). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:2)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.
An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver a iRNA agent to a tumor cell expressing αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).
A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, (β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).
Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; each of which is herein incorporated by reference.
It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.
In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.
Delivery of iRNA
The delivery of one or more iRNA to a subject in need thereof can be achieved in a number of different ways. In vivo delivery can be performed directly by administering a composition comprising an iRNA, e.g. a dsRNA, to a subject. Alternatively, delivery can be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.
Direct Delivery
In general, any method of delivering a nucleic acid molecule can be adapted for use with an iRNA (see e.g., Akhtar S, and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). However, there are three factors that are important to consider in order to successfully deliver an iRNA molecule in vivo: (a) biological stability of the delivered molecule, (2) preventing non-specific effects, and (3) accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example by direct injection or implantation into a tissue (as a non-limiting example, a tumor) or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that may otherwise be harmed by the agent or that may degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J., et al (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J., et al (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J., et al (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J., et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A., et al (2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem. 279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, JO., et al (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H., et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al (2003) J. Mol. Biol. 327:761-766; Verma, U N., et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, UN., et al (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328; Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.
Vector Encoded dsRNAs
In another aspect, iRNA targeting one or more of the EGLN genes can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as an inverted repeat joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.
iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.
iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.
Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct may be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.
Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.
Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.
In a specific embodiment, viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitates delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3:110-114 (1993). Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.
Adenoviruses are also contemplated for use in delivery of iRNAs. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10 (1994) demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT Publication WO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
Use of Adeno-associated virus (AAV) vectors is also contemplated (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Another preferred viral vector is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.
The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
III. Pharmaceutical Compositions Containing iRNA
In one embodiment, the invention provides pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical composition containing the iRNA is useful for treating a disease or disorder associated with the expression or activity of an EGLN gene, such as pathological processes mediated by EGLN expression. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) delivery. Another example is compositions that are formulated for direct delivery into the brain parenchyma, e.g., by infusion into the brain, such as by continuous pump infusion.
The pharmaceutical compositions featured herein are administered in dosages sufficient to inhibit expression of EGLN genes. In general, a suitable dose of iRNA will be in the range of 0.01 to 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The pharmaceutical composition may be administered once daily, or the iRNA may be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The effect of a single dose on EGLN levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as pathological processes mediated by EGLN expression. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. A suitable mouse model is, for example, a mouse containing a transgene expressing human EGLN.
The present invention also includes pharmaceutical compositions and formulations that include the iRNA compounds featured in the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.
The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).
Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs may be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.
Liposomal Formulations
There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the composition to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.
In order to traverse intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.
Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. There is growing evidence that for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight DNA into the skin. Compounds including analgesics, antibodies, hormones and high-molecular weight DNAs have been administered to the skin. The majority of applications resulted in the targeting of the upper epidermis
Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged DNA molecules to form a stable complex. The positively charged DNA/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some DNA is entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 1992, 19, 269-274).
One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal drug formulations to the skin. Application of liposomes containing interferon to guinea pig skin resulted in a reduction of skin herpes sores while delivery of interferon via other means (e.g., as a solution or as an emulsion) were ineffective (Weiner et al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an additional study tested the efficacy of interferon administered as part of a liposomal formulation to the administration of interferon using an aqueous system, and concluded that the liposomal formulation was superior to aqueous administration (du Plessis et al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporin-A into different layers of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).
Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).
Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (Biochimica et Biophysica Acta, 1990, 1029, 91) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0 445 131 B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496 813 B1). Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.
A number of liposomes comprising nucleic acids are known in the art. WO 96/40062 to Thierry et al. discloses methods for encapsulating high molecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomes and asserts that the contents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods of encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. discloses liposomes comprising dsRNAs targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes may be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
Nucleic Acid Lipid Particles
In one embodiment, an EGLN dsRNA featured in the invention is fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.
In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid may comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.
In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.
In one embodiment, the lipid-siRNA particle includes 40% 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio.
The non-cationic lipid may be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.
The conjugated lipid that inhibits aggregation of particles may be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmityloxypropyl (Ci6), or a PEG-distearyloxypropyl (C]8). The conjugated lipid that prevents aggregation of particles may be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.
In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.
LNP01
In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is herein incorporated by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.
Additional exemplary lipid-dsRNA formulations are as follows:
cationic lipid/non-cationic
lipid/cholesterol/PEG-lipid conjugate
Cationic Lipid
Lipid:siRNA ratio
SNALP
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-
dimethylaminopropane (DLinDMA)
cDMA
(57.1/7.1/34.4/1.4)
lipid:siRNA ~7:l
S-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DPPC/Cholesterol/PEG-cDMA
[1,3]-dioxolane (XTC)
57.1/7.1/34.4/1.4
lipid:siRNA ~7:1
LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA ~6:1
LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
57.5/7.5/31.5/3.5
lipid:siRNA ~11:1
LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA ~6:1
LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
60/7.5/31/1.5,
lipid:siRNA ~11:1
LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-
XTC/DSPC/Cholesterol/PEG-DMG
[1,3]-dioxolane (XTC)
50/10/38.5/1.5
Lipid:siRNA 10:1
LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-DMG
di((9Z,12Z)-octadeca-9,12-
50/10/38.5/1.5
dienyl)tetrahydro-3aH-
Lipid:siRNA 10:1
cyclopenta[d][1,3]-dioxol-5-amine
(ALN100)
LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-
MC-3/DSPC/Cholesterol/PEG-DMG
6,9,28,31-tetraen-19-yl 4-
50/10/38.5/1.5
(dimethylamino)butanoate (MC3)
Lipid:siRNA 10:1
LNP12
1,1′-(2-(4-(2-((2-(bis(2-
C12-200/DSPC/Cholesterol/PEG-DMG
hydroxydodecyl)amino)ethyl)(2-
50/10/38.5/1.5
hydroxydodecyl)amino)ethyl)piperazin-
Lipid:siRNA 10:1
1-yl)ethylazanediyl)didodecan-2-ol
(C12-200)
LNP13
XTC
XTC/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 33:1
LNP14
MC3
MC3/DSPC/Chol/PEG-DMG
40/15/40/5
Lipid:siRNA: 11:1
LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-
PEG-DSG
50/10/35/4.5/0.5
Lipid:siRNA: 11:1
LNP16
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP17
MC3
MC3/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP18
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/38.5/1.5
Lipid:siRNA: 12:1
LNP19
MC3
MC3/DSPC/Chol/PEG-DMG
50/10/35/5
Lipid:siRNA: 8:1
LNP20
MC3
MC3/DSPC/Chol/PEG-DPG
50/10/38.5/1.5
Lipid:siRNA: 10:1
LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 7:1
LNP22
XTC
XTC/DSPC/Chol/PEG-DSG
50/10/38.5/1.5
Lipid:siRNA: 10:1
DSPC: distearoylphosphatidylcholine
DPPC: dipalmitoylphosphatidylcholine
PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)
PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)
PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)
SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference.
XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference.
MC3 comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No. 61/185,800, filed Jun. 10, 2009, and International Application No. PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated by reference.
ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference.
C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.
Synthesis of Cationic Lipids
Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention may be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.
“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, ten-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.
“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.
“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.
“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.
“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle may be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.
The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents may be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.
“Halogen” means fluoro, chloro, bromo and iodo.
In some embodiments, the methods of the invention may require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.
Synthesis of Formula A
In one embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:
where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above may be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.
Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.
Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.
Synthesis of MC3
Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).
Synthesis of ALNY-100
Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:
Synthesis of 515:
To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na2SO4 solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g 1H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).
Synthesis of 516:
To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO3 solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27(m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60(m, 2H), 2.30-2.25(m, 2H). LC-MS [M+H] −232.3 (96.94%).
Synthesis of 517A and 517B:
The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na2SO3 and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50 mL). Organic phase was dried over an.Na2SO4 and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield: −6 g crude
517A-Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31(m, 5H), 5.04(s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47(d, 2H), 3.94-3.93(m, 2H), 2.71(s, 3H), 1.72-1.67(m, 4H). LC-MS-[M+H]−266.3, [M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.
Synthesis of 518:
Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33(m, 4H), 7.30-7.27(m, 1H), 5.37-5.27(m, 8H), 5.12(s, 2H), 4.75(m, 1H), 4.58-4.57(m, 2H), 2.78-2.74(m, 7H), 2.06-2.00(m, 8H), 1.96-1.91(m, 2H), 1.62(m, 4H), 1.48(m, 2H), 1.37-1.25(br m, 36H), 0.87(m, 6H). HPLC-98.65%.
General Procedure for the Synthesis of Compound 519:
A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na2SO4 then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (x2), 127.9 (x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6 (x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc. 654.6. Found 654.6.
Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano Z S (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.
Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.
Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.
The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
Additional Formulations
Emulsions
The compositions of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.
In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.
Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.
Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
Penetration Enhancers
In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs may cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.
Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.
Surfactants: In connection with the present invention, surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).
Bile salts: The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).
Non-chelating Non-surfactants: As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of iRNAs at the cellular level may also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTERT™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.
Other agents may be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
Carriers
Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.
Excipients
In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).
Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions may also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.
In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more biologic agents which function by a non-RNAi mechanism.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured in the invention lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by EGLN expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of an EGLN Gene
The invention relates in particular to the use of an iRNA targeting EGLN and compositions containing at least one such iRNA for the treatment of an EGLN-mediated disorder or disease. For example, a composition containing an iRNA targeting at least one EGLN gene is used for treatment of anemia. As used herein, “anemia” refers to a condition whereby the body has fewer than necessary red blood cells thereby resulting in reduced oxygen to cells and tissues. Anemias may be caused by any of several disorders and include, but are not limited to anemia due to B12 deficiency, anemia due to folate deficiency, anemia due to iron deficiency, hemolytic anemia, hemolytic anemia due to G-6-PD deficiency, idiopathic aplastic anemia, idiopathic autoimmune hemolytic anemia, immune hemolytic anemia, iegaloblastic anemia, pernicious anemia, secondary aplastic anemia, and sickle cell anemia. Certain symptoms are associated with anemia and include pale skin, dizziness, fatigue, headaches, irritability, low body temperature, numb/cold hands or feet, rapid heartbeat, shortness of breath, weakness and chest pain any of which may be ameliorated by administration of the iRNA agents targeting one or more EGLN genes of the present invention.
In one embodiment at least one iRNA targeting at least one EGLN gene is used to downregulate hepcidin (GenBank Reference NG—011563.1; SEQ ID 2805 representing the complete gene on chromosome 19; and GenBank Reference NM—021175 representing the Hepcidin peptide; SEQ ID NO: 2806). Probes for the detection of hepcidin (HAMP1) were purchased from Panomics (a division of Affymetrix, Santa Clara, Calif.) and can detect either HAMP1 or HAMP2. Hepcidin is a peptide hormone that is produced by the liver. It is believed that hepcidin binds to ion channel to inhibit iron transport out of the cells which store iron. The downregulation of hepcidin may result in increased mobilization of iron in the body.
In one embodiment at least one iRNA targeting at least one EGLN gene is used for the treatment of cancer. As used herein “cancer” refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. A cancer can be a tumor or hematological malignancy, and includes but is not limited to, all types of cancers but preferably leukemias, and those arising in the blood or bone.
Leukemias, or cancers of the blood or bone marrow that are characterized by an abnormal proliferation of white blood cells i.e., leukocytes, can be divided into four major classifications including Acute lymphoblastic leukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenous leukemia or acute myeloid leukemia (AML) (AML with translocations between chromosome 10 and 11 [t(10, 11)], chromosome 8 and 21 [t(8;21)], chromosome 15 and 17 [t(15;17)], and inversions in chromosome 16 [inv(16)]; AML with multilineage dysplasia, which includes patients who have had a prior myelodysplastic syndrome (MDS) or myeloproliferative disease that transforms into AML; AML and myelodysplastic syndrome (MDS), therapy-related, which category includes patients who have had prior chemotherapy and/or radiation and subsequently develop AML or MDS; d) AML not otherwise categorized, which includes subtypes of AML that do not fall into the above categories; and e) Acute leukemias of ambiguous lineage, which occur when the leukemic cells can not be classified as either myeloid or lymphoid cells, or where both types of cells are present); and Chronic myelogenous leukemia (CML). These types of leukemias are particularly amenable to treatment with the iRNA agents of the present invention.
The invention further relates to the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating anemia or cancer, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, the iRNA or pharmaceutical composition thereof can also be administered in conjunction with one or more additional anti-cancer treatments, such as biological, chemotherapy and radiotherapy. Accordingly, a treatment can include, for example, imatinib (Gleevac), all-trans-retinoic acid, a monoclonal antibody treatment (gemtuzumab, ozogamicin), chemotherapy (for example, chlorambucil, prednisone, prednisolone, vincristine, cytarabine, clofarabine, farnesyl transferase inhibitors, decitabine, inhibitors of MDR1), rituximab, interferon-α, anthracycline drugs (such as daunorubicin or idarubicin), L-asparaginase, doxorubicin, cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide, pentostatin, or cladribine), bone marrow transplant, stem cell transplant, radiation thereapy, anti-metabolite drugs (methotrexate and 6-mercaptopurine), or any combination thereof.
In one embodiment, the iRNA agents of the present invention may be administered in combination with an iron supplement. The administration may be simultaneously, together, or apart. The dosing may be on the same schedule, an offset schedule or a one time administration of the iron supplement. The iron supplement may be given on an “as needed” basis depending on measurements made in the particular patient.
Radiation therapy (also called radiotherapy, X-ray therapy, or irradiation) is the use of ionizing radiation to kill cancer cells and shrink tumors. Radiation therapy can be administered externally via external beam radiotherapy (EBRT) or internally via brachytherapy. The effects of radiation therapy are localised and confined to the region being treated. Radiation therapy may be used to treat almost every type of solid tumor, including cancers of the brain, breast, cervix, larynx, lung, pancreas, prostate, skin, stomach, uterus, or soft tissue sarcomas. Radiation is also used to treat leukemia and lymphoma.
Chemotherapy is the treatment of cancer with drugs that can destroy cancer cells. In current usage, the term “chemotherapy” usually refers to cytotoxic drugs which affect rapidly dividing cells in general, in contrast with targeted therapy. Chemotherapy drugs interfere with cell division in various possible ways, e.g. with the duplication of DNA or the separation of newly formed chromosomes. Most forms of chemotherapy target all rapidly dividing cells and are not specific to cancer cells, although some degree of specificity may come from the inability of many cancer cells to repair DNA damage, while normal cells generally can. Most chemotherapy regimens are given in combination. Exemplary chemotherapeutic agents include, but are not limited to, 5-FU Enhancer, 9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide, Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY 12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine, Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors, Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl, D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel, Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR, Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide (VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol, Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline, Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2, Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate (LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gamma linolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat, Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, Meglamine GLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone, Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698, OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARP Inhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412, Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS Farnesyl Transferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU), Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide, Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase, UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesine sulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD 9331.
Biological therapies use the body's immune system, either directly or indirectly, to fight cancer or to lessen the side effects that may be caused by some cancer treatments. In one sense, targeting one or more EGLN genes can be considered in this group of therapies in that it can stimulate immune system action against a tumor, for example. However, this approach can also be considered with other such biological approaches, e.g., immune response modifying therapies such as the administration of interferons, interleukins, colony-stimulating factors, monoclonal antibodies, vaccines, gene therapy, and nonspecific immunomodulating agents are also envisioned as anti-cancer therapies to be combined with the inhibition of EGLN. Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell, such as tyrosine kinase inhibitors imatinib (Gleevec/Glivec) and gefitinib (Iressa). Examples of monoclonal antibody therapies that can be used with an iRNA or pharmaceutical composition thereof include, but are not limited to, the anti-HER2/neu antibody trastuzumab (Herceptin) used in breast cancer, and the anti-CD20 antibody rituximab, used in a variety of B-cell malignancies. The growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancers. Removing or blocking estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens may be therapeutically beneficial.
Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the patient's own immune system to fight the tumor, and include, but are not limited to, intravesical BCG immunotherapy for superficial bladder cancer, vaccines to generate specific immune responses, such as for malignant melanoma and renal cell carcinoma, and the use of Sipuleucel-T for prostate cancer, in which dendritic cells from the patient are loaded with prostatic acid phosphatase peptides to induce a specific immune response against prostate-derived cells.
In some embodiments, an iRNA targeting one or more EGLN genes is administered in combination with an angiogenesis inhibitor. In some embodiments, the angiogenesis inhibitors for use in the methods described herein include, but are not limited to, monoclonal antibody therapies directed against specific pro-angiogenic growth factors and/or their receptors. Examples of these are: bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix™), and trastuzumab (Herceptin®). In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to small molecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor receptors. The three TKIs that are currently approved as anti-cancer therapies are erlotinib (Tarceva®), sorafenib (Nexavar®), and sunitinib (Sutent®). In some embodiments, the angiogenesis inhibitors for use in the methods described herein include but are not limited to inhibitors of mTOR (mammalian target of rapamycin) such as temsirolimus (Toricel™), bortezomib (Velcade®), thalidomide (Thalomid®), and Doxycyclin.
In other embodiments, the angiogenesis inhibitors for use in the methods described herein include one or more drugs that target the VEGF pathway, including, but not limited to, Bevacizumab (Avastin®), sunitinib (Sutent®), and sorafenib (Nexavar®). Additional VEGF inhibitors include CP-547,632 (3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin 1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride; Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474 (AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca), CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788 (Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; Bayer Pharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known as PTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptanib octasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 (glufanide disodium, Cytran Inc. of Kirkland, Wash., USA), VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.), angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron (Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor, Sirna Therapeutics, San Francisco, Calif.) Caplostatin, soluble ectodomains of the VEGF receptors, Neovastat (1Eterna Zentaris Inc; Quebec City, Calif.), ZM323881 (CalBiochem. CA, USA), pegaptanib (Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer and combinations thereof.
In other embodiments, the angiogenesis inhibitors for use in the methods described herein include anti-angiogenic factors such as alpha-2 antiplasmin (fragment), angiostatin (plasminogen fragment), antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59 complement fragment, endostatin (collagen XVIII fragment), fibronectin fragment, gro-beta (a C—X—C chemokine), heparinases heparin hexasaccharide fragment, human chorionic gonadotropin (hCG), interferon alpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12, kringle 5 (plasminogen fragment), beta-thromboglobulin, EGF (fragment), VEGF inhibitor, endostatin, fibronection (45 kD fragment), high molecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF, PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1, prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs), 2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogen activator inhibitor, prolactin 16 kD fragment, proliferin-related protein (PRP), retinoids, tetrahydrocortisol-S transforming growth factor-beta (TGF-b), vasculostatin, and vasostatin (calreticulin fragment).pamidronate thalidomide, TNP470, the bisphosphonate family such as amino-bisphosphonate zoledronic acid. bombesin/gastrin-releasing peptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol A M, et. al., British Journal of Cancer (2004) 90, 245-252), anti-VEGF peptide (dRK6) (Seung-Ah Yoo, J. Immuno, 2005, 174: 5846-5855).
Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting one or more EGLN genes or pharmaceutical composition thereof, “effective against” a cancer indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.
In one embodiment the disorder is anemia where efficacy of treatment can be determined by measuring standard endpoints associated with improvement anemia due to B12 deficiency, anemia due to folate deficiency, anemia due to iron deficiency, hemolytic anemia, hemolytic anemia due to G-6-PD deficiency, idiopathic aplastic anemia, idiopathic autoimmune hemolytic anemia, immune hemolytic anemia, iegaloblastic anemia, pernicious anemia, secondary aplastic anemia, and sickle cell anemia. For example, an improvement in any of the manifestations of anemia such as pale skin, dizziness, fatigue, headaches, irritability, low body temperature, numb/cold hands or feet, rapid heartbeat, reduced erythropoietin, shortness of breath, weakness and chest pain would be considered indicative of effective treatment.
A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.
The invention relates in particular to the use of one or more iRNA targeting one or more EGLN genes and compositions containing at least one such iRNA for the treatment of an EGLN-mediated disorder or disease. For example, a composition containing an iRNA targeting an EGLN gene is used for treatment of an infectious disease or disorder, for example, in a subject having an infection. In some preferred embodiments the subject has an infection or is at risk of having an infection. An “infection” as used herein refers to a disease or condition attributable to the presence in a host of a foreign organism or agent that reproduces within the host. Infections typically involve breach of a normal mucosal or other tissue barrier by an infectious organism or agent. A subject that has an infection is a subject having objectively measurable infectious organisms or agents present in the subject's body. A subject at risk of having an infection is a subject that is predisposed to develop an infection. Such a subject can include, for example, a subject with a known or suspected exposure to an infectious organism or agent. A subject at risk of having an infection also can include a subject with a condition associated with impaired ability to mount an immune response to an infectious organism or agent, e.g., a subject with a congenital or acquired immunodeficiency, a subject undergoing radiation therapy or chemotherapy, a subject with a burn injury, a subject with a traumatic injury, a subject undergoing surgery or other invasive medical or dental procedure.
Infections are broadly classified as bacterial, viral, fungal, or parasitic based on the category of infectious organism or agent involved. Other less common types of infection are also known in the art, including, e.g., infections involving rickettsiae, mycoplasmas, and agents causing scrapie, bovine spongiform encephalopthy (BSE), and prion diseases (e.g., kuru and Creutzfeldt-Jacob disease). Examples of bacteria, viruses, fungi, and parasites which cause infection are well known in the art. An infection can be acute, subacute, chronic, or latent, and it can be localized or systemic. As defined herein, a “chronic infection” refers to those infections that are not cleared by the normal actions of the innate or adaptive immune responses and persist in the subject for a long duration of time, on the order of weeks, months, and years. A chronic infection may reflect latency of the infectious agent, and may be include periods in which no infectious symptoms are present, i.e., asymptomatic periods. Examples of chronic infections include, but are not limited to, HIV infection and herpesvirus infections. Furthermore, an infection can be predominantly intracellular or extracellular during at least one phase of the infectious organism's or agent's life cycle in the host.
Exemplary viruses include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III), HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses, i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae; Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus; Rous sarcoma virus; West Nile virus; Japanese equine encephalitis, Norwalk, papilloma virus, parvovirus B19; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=enterally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).
Bacteria include both Gram negative and Gram positive bacteria. Examples of Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Examples of Gram negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae, M. leprae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter spp., Enterococcus spp., Haemophilus influenzae (Hemophilus influenza B, and Hemophilus influenza non-typable), Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelii, meningococcus, pertussis, pneumococcus, shigella, tetanus, Vibrio cholerae, yersinia, Pseudomonas species, Clostridia species, Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia, Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus, Pseudomonas aeruginosa, Cryptosporidium parvum, Streptococcus pneumoniae, and Bordetella pertussis.
Exemplary fungi and yeast include, but are not limited to, Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Blastomyces dermatitidis, Aspergillus clavatus, Cryptococcus neoformans, Chlamydia trachomatis, Coccidioides immitis, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Nocardia spp, Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotrys chartarum, and any combination thereof.
Exemplary parasites include, but are not limited to: Entamoeba histolytica; Plasmodium species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax), Leishmania species (Leishmania tropica, Leishmania braziliensis, Leishmania donovani), Toxoplasmosis (Toxoplasma gondii), Trypanosoma gambiense, Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), Helminths (flat worms, round worms), Babesia microti, Babesia divergens, Giardia lamblia, and any combination thereof.
The invention further relates to the use of an iRNA targeting one or more EGLN genes and compositions containing at least one such iRNA for the treatment of an infectious disease, such as hepatitis B or a chronic bacterial infection, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating such infectious diseases or disorders (e.g., antibiotics, anti-viral agents). For example, in certain embodiments, administration of one or more dsRNA targeting EGLN is administered in combination with an antibacterial agent. Examples of anti-bacterial agents useful for the methods described herein include, but are not limited to, natural penicillins, semi-synthetic penicillins, clavulanic acid, cephalolsporins, bacitracin, ampicillin, carbenicillin, oxacillin, azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin, nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor, cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet, cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam, carbapenems, imipenems, monobactems, eurtreonam, vancomycin, polymyxin, amphotericin B, nystatin, imidazoles, clotrimazole, miconazole, ketoconazole, itraconazole, fluconazole, rifampins, ethambutol, tetracyclines, chloramphenicol, macrolides, aminoglycosides, streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline, minocycline, doxycycline, chlortetracycline, erythromycin, roxithromycin, clarithromycin, oleandomycin, azithromycin, chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin, enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, and trimethoprim; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine; Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; Amifloxacin Mesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid; Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; Ampicillin Sodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate; Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium; Bacampicillin Hydrochloride; Bacitracin; Bacitracin Methylene Disalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium; Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; Biphenamine Hydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate; Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; Carbenicillin Indanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium; Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate; Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium; Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; Cefepime Hydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride; Cefinetazole; Cefinetazole Sodium; Cefonicid Monosodium; Cefonicid Sodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan; Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium; Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium; Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine; Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium; Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil; Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; Cephalexin Hydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium; Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol; Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol Pantothenate Complex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate; Chloroxylenol; Chlortetracycline Bisulfate; Chlortetracycline Hydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride; Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin; Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride; Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; Cloxacillin Sodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin; Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone; Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline; Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium; Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline; Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; Droxacin Sodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride; Erythromycin; Erythromycin Acistrate; Erythromycin Estolate; Erythromycin Ethylsuccinate; Erythromycin Gluceptate; Erythromycin Lactobionate; Erythromycin Propionate; Erythromycin Stearate; Ethambutol Hydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine; Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin; Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid; Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin; Hetacillin Potassium; Hexedine; Ibafloxacin; Inipenem; Isoconazole; Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin; Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin; Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride; Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; Meclocycline Sulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem; Methacycline; Methacycline Hydrochloride; Methenamine; Methenamine Hippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim; Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin; Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; Mirincamycin Hydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; Nalidixate Sodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate; Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate; Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone; Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole; Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium; Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium; Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; Oxytetracycline Hydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin; Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin G Potassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V; Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin V Potassium; Pentizidone Sodium; Phenyl Aminosalicylate; Piperacillin Sodium; Pirbenicillin Sodium; Piridicillin Sodium; Pirlimycin Hydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate; Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin; Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin; Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin; Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin; Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; Rosaramicin Butyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate; Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline; Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin; Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride; Spiramycin; Stallimycin Hydrochloride; Steffimycin; Streptomycin Sulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide; Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium; Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine; Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole; Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole; Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl; Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; Suncillin Sodium; Talampicillin Hydrochloride; Teicoplanin; Temafloxacin Hydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride; Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol; Thiphencillin Potassium; Ticarcillin Cresyl Sodium; Ticarcillin Disodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride; Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; Trimethoprim Sulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate; Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; and Zorbamycin.
In other embodiments, administration of one or more dsRNA targeting one or more EGLN genes is performed in combination with an anti-viral medicament or agent. Exemplary antiviral agents useful for the methods described herein include, but are not limited to, immunoglobulins, amantadine, interferon, nucleoside analogues, and protease inhibitors. Specific examples of antiviral agents include but are not limited to Acemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride; Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium; Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine; Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine; Zidovudine; and Zinviroxime.
In other embodiments, administration of one or more dsRNA targeting one or more EGLN genes is performed in combination with an anti-fungal medicament or agent. An “antifungal medicament” is an agent that kills or inhibits the growth or function of infective fungi. Anti-fungal medicaments are sometimes classified by their mechanism of action. Some anti-fungal agents function as cell wall inhibitors by inhibiting glucose synthase, other antifungal agents function by destabilizing membrane integrity, and other antifungal agents function by breaking down chitin (e.g., chitinase) or immunosuppression (501 cream). Thus, exemplary antifungal medicaments useful for the methods described herein include, but are not limited to, imidazoles, 501 cream, and Acrisorcin, Ambruticin, Amorolfine, Amphotericin B, Azaconazole, Azaserine, Basifungin, BAY 38-9502, Bifonazole, Biphenamine Hydrochloride, Bispyrithione Magsulfex, Butenafine, Butoconazole Nitrate, Calcium Undecylenate, Candicidin, Carbol-Fuchsin, Chitinase, Chlordantoin, Ciclopirox, Ciclopirox Olamine, Cilofungin, Cisconazole, Clotrimazole, Cuprimyxin, Denofungin, Dipyrithione, Doconazole, Econazole, Econazole Nitrate, Enilconazole, Ethonam Nitrate, Fenticonazole Nitrate, Filipin, FK 463, Fluconazole, Flucytosine, Fungimycin, Griseofulvin, Hamycin, Isoconazole, Itraconazole, Kalafungin, Ketoconazole, Lomofungin, Lydimycin, Mepartricin, Miconazole, Miconazole Nitrate, MK 991, Monensin, Monensin Sodium, Naftifine Hydrochloride, Neomycin Undecylenate, Nifuratel, Nifurmerone, Nitralamine Hydrochloride, Nystatin, Octanoic Acid, Orconazole Nitrate, Oxiconazole Nitrate, Oxifungin Hydrochloride, Parconazole Hydrochloride, Partricin, Potassium Iodide, Pradimicin, Proclonol, Pyrithione Zinc, PyrroInitrin, Rutamycin, Sanguinarium Chloride, Saperconazole, Scopafungin, Selenium Sulfide, Sertaconazole, Sinefungin, Sulconazole Nitrate, Terbinafine, Terconazole, Thiram, Ticlatone, Tioconazole, Tolciclate, Tolindate, Tolnaftate, Triacetin, Triafungin, UK 292, Undecylenic Acid, Viridofulvin, Voriconazole, Zinc Undecylenate, and Zinoconazole Hydrochloride.
In further embodiments, administration of one or more dsRNA targeting one or more EGLN genes is administered in combination with an anti-parasitic medicament or agent. An “antiparasitic medicament” refers to an agent that kills or inhibits the growth or function of infective parasites. Examples of antiparasitic medicaments, also referred to as parasiticides, useful for the methods described herein include, but are not limited to, albendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine, diloxanide furoate, doxycycline, eflomithine, furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumine antimoniate, melarsoprol, metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine, paromomycin, pentamidine isethionate, piperazine, praziquantel, primaquine phosphate, proguanil, pyrantel pamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium (sodium antimony gluconate), suramin, tetracycline, thiabendazole, timidazole, trimethroprim-sulfamethoxazole, and tryparsamide, some of which are used alone or in combination with others.
The iRNA and an additional therapeutic agent can be administered in combination in the same composition, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or by another method described herein.
Patients can be administered a therapeutic amount of iRNA, such as 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The iRNA can be administered by intravenous infusion over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The administration is repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce EGLN levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.
Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction, or for elevated lipid levels or blood pressure. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.
Genetic predisposition plays a role in the development of some cancers and hematological malignancies. Therefore, a patient in need of one or more EGLN iRNA may be identified by taking a family history, or, for example, screening for one or more genetic markers or variants. A healthcare provider, such as a doctor, nurse, or family member, can take a family history before prescribing or administering an EGLN dsRNA. For example, certain variants in the BRCA1 and BRCA2 genes are known to cause an increased risk for breast and ovarian cancers. A DNA test may also be performed on the patient to identify a mutation in an EGLN gene, before an EGLN dsRNA is administered to the patient.
Owing to the inhibitory effects on EGLN expression, a composition according to the invention or a pharmaceutical composition prepared therefrom can enhance the quality of life.
Methods for Modulating Expression of an EGLN Gene
In yet another aspect, the invention provides a method for modulating (e.g., inhibiting or activating) the expression of an EGLN gene in a mammal
In one embodiment, the method includes administering a composition featured in the invention to the mammal such that expression of the target EGLN gene is decreased, such as for an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, or four weeks or longer.
In another embodiment, the method includes administering a composition as described herein to a mammal such that expression of the target EGLN gene is increased by e.g., at least 10% compared to an untreated animal. In some embodiments, the activation of EGLN occurs over an extended duration, e.g., at least two, three, four days or more, e.g., one week, two weeks, three weeks, four weeks, or more. Without wishing to be bound by theory, an iRNA can activate EGLN expression by stabilizing an EGLN mRNA transcript, interacting with a promoter in the genome, and/or inhibiting an inhibitor of EGLN expression.
Preferably, the iRNAs useful for the methods and compositions featured in the invention specifically target RNAs (primary or processed) of the target EGLN gene. Compositions and methods for inhibiting the expression of these EGLN genes using iRNAs can be prepared and performed as described elsewhere herein.
In one embodiment, the method includes administering a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of an EGLN gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection.
In one embodiment iRNAs are able to substantially target a single organ of the body. The targeted organ may be, but is not limited to, the liver, kidney and spleen. In another embodiment, the organ substantially targeted is the liver.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
iRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
Oligonucleotide Synthesis.
All oligonucleotides are synthesized on an AKTAoligopilot synthesizer. Commercially available controlled pore glass solid support (dT-CPG, 500, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5′-O-dimethoxytrityl N6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, 5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite, and 5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis. The 2′-F phosphoramidites, 5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite and 5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramidite are purchased from (Promega). All phosphoramidites are used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which is used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes is used. The activator is 5-ethyl thiotetrazole (0.75M, American International Chemicals); for the PO-oxidation iodine/water/pyridine is used and for the PS-oxidation PADS (2%) in 2,6-lutidine/ACN (1:1 v/v) is used.
3′-ligand conjugated strands are synthesized using solid support containing the corresponding ligand. For example, the introduction of cholesterol unit in the sequence is performed from a hydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered to trans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain a hydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore) labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3) phosphoramidite are purchased from Biosearch Technologies. Conjugation of ligands to 5′-end and or internal position is achieved by using appropriately protected ligand-phosphoramidite building block. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator to a solid-support-bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate is carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate is introduced by the oxidation of phosphite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent. The cholesterol phosphoramidite is synthesized in house and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite is 16 minutes.
Deprotection I (Nucleobase Deprotection)
After completion of synthesis, the support is transferred to a 100 mL glass bottle (VWR). The oligonucleotide is cleaved from the support with simultaneous deprotection of base and phosphate groups with 80 mL of a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C. The bottle is cooled briefly on ice and then the ethanolic ammonia mixture is filtered into a new 250-mL bottle. The CPG is washed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixture is then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dry ice and dried under vacuum on a speed vac.
Deprotection II (Removal of 2′-TBDMS Group)
The dried residue is resuspended in 26 mL of triethylamine, triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60° C. for 90 minutes to remove the tert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reaction is then quenched with 50 mL of 20 mM sodium acetate and the pH is adjusted to 6.5. Oligonucleotide is stored in a freezer until purification.
Analysis
The oligonucleotides are analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated ligand.
HPLC Purification
The ligand-conjugated oligonucleotides are purified by reverse-phase preparative HPLC. The unconjugated oligonucleotides are purified by anion-exchange HPLC on a TSK gel column packed in house. The buffers are 20 mM sodium phosphate (pH 8.5) in 10% CH3CN (buffer A) and 20 mM sodium phosphate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing full-length oligonucleotides are pooled, desalted, and lyophilized. Approximately 0.15 OD of desalted oligonucleotidess are diluted in water to 150 μL and then pipetted into special vials for CGE and LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.
iRNA Preparation
For the general preparation of iRNA, equimolar amounts of sense and antisense strand are heated in 1×PBS at 95° C. for 5 min and slowly cooled to room temperature. Integrity of the duplex is confirmed by HPLC analysis.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 1.
TABLE 1
Abbreviations of nucleotide monomers used
in nucleic acid sequence representation. It will
be understood that these monomers, when
present in an oligonucleotide, are mutually
linked by 5′-3′-phosphodiester bonds.
Abbreviation
Nucleotide(s)
A
Adenosine
C
Cytidine
G
Guanosine
T
Thymidine
U
Uridine
N
any nucleotide (G, A, C, T or U)
a
2′-O-methyladenosine
c
2′-O-methylcytidine
g
2′-O-methylguanosine
u
2′-O-methyluridine
dT
2′-deoxythymidine
s
phosphorothioate linkage
Example 2
EGLN siRNA Design and Synthesis
Transcripts
Oligonucleotide design was carried out to identify siRNAs targeting the genes encoding the mouse (Mus musculus) EGLN 1, 2 and 3 genes. The design process used the EGLN transcript NM—053207.2 for EGLN1 (SEQ ID NO: 5), NM—053208.4 for EGLN2 (SEQ ID NO: 6), and NM—028133.2 for EGLN3 (SEQ ID NO: 7). All sequences were obtained from the NCBI Refseq collection.
The orthologous sequences from humans (Homo sapiens) were also designed. Oligonucleotide design was carried out to identify siRNAs targeting the genes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. The design process used the EGLN transcript NM—022051.2 for EGLN1 (SEQ ID NO: 390), NM—053046.2 for EGLN2 (SEQ ID NO: 391), and NM—022073.3 for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBI Refseq collection.
The set of mouse EGLN derived siRNA oligos designed and synthesized are presented in Tables 2A-F.
The set of human EGLN derived siRNA oligonucleotide single and double strand duplexes designed are presented in Tables 6A-C.
siRNA Design and Specificity Prediction
The specificity of the 19mer oligo sets was predicted from each sequence. The EGLN siRNAs were used in a comprehensive search against their respective human, or mouse and rat transcriptomes (defined as the set of NM_ and XM_records within the NCBI Refseq set) using the FASTA algorithm. The Python script ‘offtargetFasta.py’ was then used to parse the alignments and generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. The off-target score is calculated as follows: mismatches between the oligo and the transcript are given penalties. A mismatch in the seed region in positions 2-9 of the oligo is given a penalty of 2.8; mismatches in the putative cleavage sites 10 and 11 are given a penalty of 1.2, and all other mismatches a penalty of 1. The off-target score for each oligo-transcript pair is then calculated by summing the mismatch penalties. The lowest off-target score from all the oligo-transcript pairs is then determined and used in subsequent sorting of oligos. Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderate specific. In picking which oligos to synthesize, we sorted from high to low by the off-target score of the antisense strand and took the best (lowest off-target score) oligo pairs.
Synthesis of EGLN Sequences
EGLN targeting sequences were synthesized on a MerMade 192 synthesizer at 1 μmol scale.
For all chemically modified sequences in the list, ‘endolight’ chemistry was applied as detailed below.
All pyrimidines (cytosine and uridine) in the sense strand contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)
In the antisense strand, pyrimidines adjacent to(towards 5′ position) ribo A nucleoside were replaced with their corresponding 2-O-Methyl nucleosides
A two base dTsdT extension at 3′ end of both sense and antisense sequences was introduced
The sequence file was converted to a text file to make it compatible for loading in the MerMade 192 synthesis software
Synthesis, Cleavage and Deprotection:
The synthesis of EGLN sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry.
The synthesis of the above sequences was performed at 1 um scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.
The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. The crude sequences were precipitated using acetone:ethanol (80:20) mix and the pellet were re-suspended in 0.02M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.
Purification and Desalting:
EGLN sequences were purified on AKTA explorer purification system using Source 15Q column. A column temperature of 65 C was maintained during purification. Sample injection and collection was performed in 96 well (1.8 mL-deep well) plates. A single peak corresponding to the full length sequence was collected in the eluent. The purified sequences were desalted on a Sephadex G25 column using AKTA purifier. The desalted EGLN sequences were analyzed for concentration (by UV measurement at A260) and purity (by ion exchange HPLC). The single strands were then submitted for annealing. The control duplex, AD-1955, which targets the luciferase gene has the sense sequence cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 8) and the antisense sequence UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 9), where lower case nucleotides are modified by 2′Omethyl and dT stands for deoxyThymidine and “s” represents a phosphorothioate linkage.
TABLE 2A
Mouse EGNL1 Single Strands and Duplex Sequences
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-40893
1057
GCUAUGUCCGUCACGUUGA
10
UCAACGUGACGGACAUAGC
11
AD-40899
1065
CGUCACGUUGAUAACCCAA
12
UUGGGUUAUCAACGUGACG
13
AD-40905
1092
GGAAGAUGCGUGACAUGUA
14
UACAUGUCACGCAUCUUCC
15
AD-40911
1128
GACUGGGACGCCAAGGUAA
16
UUACCUUGGCGUCCCAGUC
17
AD-40917
1150
GAGGUAUUCUUCGAAUUUU
18
AAAAUUCGAAGAAUACCUC
19
AD-40923
1240
GGCGUAACCCUCAUGAAGU
20
ACUUCAUGAGGGUUACGCC
21
AD-40929
1271
CGCCACAAGGUACGCAAUA
22
UAUUGCGUACCUUGUGGCG
23
AD-40888
1272
GCCACAAGGUACGCAAUAA
24
UUAUUGCGUACCUUGUGGC
25
AD-40894
1276
CAAGGUACGCAAUAACUGU
26
ACAGUUAUUGCGUACCUUG
27
AD-40900
1317
CGAGCGAGAGCUAAAGUAA
28
UUACUUUAGCUCUCGCUCG
29
AD-40906
1320
GCGAGAGCUAAAGUAAAAU
30
AUUUUACUUUAGCUCUCGC
31
AD-40912
1356
GGUGUGAGGGUUGAACUCA
32
UGAGUUCAACCCUCACACC
33
AD-40918
1386
GUCAGCAAAGACGUCUAGU
34
ACUAGACGUCUUUGCUGAC
35
AD-40924
1892
GCCUCCUGCGAUGAUUGUU
36
AACAAUCAUCGCAGGAGGC
37
AD-40930
1919
GUGACGACGUGUUGCUUCU
38
AGAAGCAACACGUCGUCAC
39
AD-40889
2043
CGCUUCGACCGACCUAACA
40
UGUUAGGUCGGUCGAAGCG
41
AD-40895
2048
CGACCGACCUAACAGUAGA
42
UCUACUGUUAGGUCGGUCG
43
AD-40901
2093
CAACAUAGUUACAAGAGGA
44
UCCUCUUGUAACUAUGUUG
45
AD-40907
2159
CGAAGUGACGGGCACUAAA
46
UUUAGUGCCCGUCACUUCG
47
AD-40913
2160
GAAGUGACGGGCACUAAAU
48
AUUUAGUGCCCGUCACUUC
49
AD-40919
2372
GUGAAUGUGGUAUGUGGUU
50
AACCACAUACCACAUUCAC
51
AD-40925
2605
GCACAGAUUGUGGGUAUAA
52
UUAUACCCACAAUCUGUGC
53
AD-40931
2624
CUCCUGUCCCCUUAGGUGU
54
ACACCUAAGGGGACAGGAG
55
AD-40890
2732
GUUUGUAUCCGGUUAGAAA
56
UUUCUAACCGGAUACAAAC
57
AD-40896
2889
GUCUCCUUCUGACCCAUAU
58
AUAUGGGUCAGAAGGAGAC
59
AD-40902
2894
CUUCUGACCCAUAUCCGCU
60
AGCGGAUAUGGGUCAGAAG
61
AD-40908
3001
GGAACUGUUUGGCAUUGUU
62
AACAAUGCCAAACAGUUCC
63
AD-40914
3244
CUUAAUGCCCACUUAAACU
64
AGUUUAAGUGGGCAUUAAG
65
AD-40920
3272
GUUAGGACUCUUGUUUAAA
66
UUUAAACAAGAGUCCUAAC
67
AD-40926
3350
CUGUUCAACACAUUAACCA
68
UGGUUAAUGUGUUGAACAG
69
AD-40932
3472
GCUUGUAAAGCUAAUCUAA
70
UUAGAUUAGCUUUACAAGC
71
*Start is the 5′ most position on transcript NM_053207.2
TABLE 2B
Mouse EGNL1 Chemically modified Single Strands and Duplex Sequences
For all the sequences in the list, ‘endolight’ chemistry was applied as
described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-40893
1057
GcuAuGuccGucAcGuuGAdTsdT
72
UcAACGUGACGGAcAuAGCdTsdT
73
AD-40899
1065
cGucAcGuuGAuAAcccAAdTsdT
74
UUGGGUuAUcAACGUGACGdTsdT
75
AD-40905
1092
GGAAGAuGcGuGAcAuGuAdTsdT
76
uAcAUGUcACGcAUCUUCCdTsdT
77
AD-40911
1128
GAcuGGGAcGccAAGGuAAdTsdT
78
UuACCUUGGCGUCCcAGUCdTsdT
79
AD-40917
1150
GAGGuAuucuucGAAuuuudTsdT
80
AAAAUUCGAAGAAuACCUCdTsdT
81
AD-40923
1240
GGcGuAAcccucAuGAAGudTsdT
82
ACUUcAUGAGGGUuACGCCdTsdT
83
AD-40929
1271
cGccAcAAGGuAcGcAAuAdTsdT
84
uAUUGCGuACCUUGUGGCGdTsdT
85
AD-40888
1272
GccAcAAGGuAcGcAAuAAdTsdT
86
UuAUUGCGuACCUUGUGGCdTsdT
87
AD-40894
1276
cAAGGuAcGcAAuAAcuGudTsdT
88
AcAGUuAUUGCGuACCUUGdTsdT
89
AD-40900
1317
cGAGcGAGAGcuAAAGuAAdTsdT
90
UuACUUuAGCUCUCGCUCGdTsdT
91
AD-40906
1320
GcGAGAGcuAAAGuAAAAudTsdT
92
AUUUuACUUuAGCUCUCGCdTsdT
93
AD-40912
1356
GGuGuGAGGGuuGAAcucAdTsdT
94
UGAGUUcAACCCUcAcACCdTsdT
95
AD-40918
1386
GucAGcAAAGAcGucuAGudTsdT
96
ACuAGACGUCUUUGCUGACdTsdT
97
AD-40924
1892
GccuccuGcGAuGAuuGuudTsdT
98
AAcAAUcAUCGcAGGAGGCdTsdT
99
AD-40930
1919
GuGAcGAcGuGuuGcuucudTsdT
100
AGAAGcAAcACGUCGUcACdTsdT
101
AD-40889
2043
cGcuucGAccGAccuAAcAdTsdT
102
UGUuAGGUCGGUCGAAGCGdTsdT
103
AD-40895
2048
cGAccGAccuAAcAGuAGAdTsdT
104
UCuACUGUuAGGUCGGUCGdTsdT
105
AD-40901
2093
cAAcAuAGuuAcAAGAGGAdTsdT
106
UCCUCUUGuAACuAUGUUGdTsdT
107
AD-40907
2159
cGAAGuGAcGGGcAcuAAAdTsdT
108
UUuAGUGCCCGUcACUUCGdTsdT
109
AD-40913
2160
GAAGuGAcGGGcAcuAAAudTsdT
110
AUUuAGUGCCCGUcACUUCdTsdT
111
AD-40919
2372
GuGAAuGuGGuAuGuGGuudTsdT
112
AACcAcAuACcAcAUUcACdTsdT
113
AD-40925
2605
GcAcAGAuuGuGGGuAuAAdTsdT
114
UuAuACCcAcAAUCUGUGCdTsdT
115
AD-40931
2624
cuccuGuccccuuAGGuGudTsdT
116
AcACCuAAGGGGAcAGGAGdTsdT
117
AD-40890
2732
GuuuGuAuccGGuuAGAAAdTsdT
118
UUUCuAACCGGAuAcAAACdTsdT
119
AD-40896
2889
GucuccuucuGAcccAuAudTsdT
120
AuAUGGGUcAGAAGGAGACdTsdT
121
AD-40902
2894
cuucuGAcccAuAuccGcudTsdT
122
AGCGGAuAUGGGUcAGAAGdTsdT
123
AD-40908
3001
GGAAcuGuuuGGcAuuGuudTsdT
124
AAcAAUGCcAAAcAGUUCCdTsdT
125
AD-40914
3244
cuuAAuGcccAcuuAAAcudTsdT
126
AGUUuAAGUGGGcAUuAAGdTsdT
127
AD-40920
3272
GuuAGGAcucuuGuuuAAAdTsdT
128
UUuAAAcAAGAGUCCuAACdTsdT
129
AD-40926
3350
cuGuucAAcAcAuuAAccAdTsdT
130
UGGUuAAUGUGUUGAAcAGdTsdT
131
AD-40932
3472
GcuuGuAAAGcuAAucuAAdTsdT
132
UuAGAUuAGCUUuAcAAGCdTsdT
133
TABLE 2C
Mouse EGNL2 Single Strands and Duplex Sequences
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-
128
AUCAGUCCCUUCUCAAGCU
134
AGCUUGAGAAGGGACUGAU
135
40891
AD-
418
GUCCUUGGAGUCUAGCCGA
136
UCGGCUAGACUCCAAGGAC
137
40897
AD-
545
GCCACUGCUACUACGACCA
138
UGGUCGUAGUAGCAGUGGC
139
40903
AD-
934
GCCUUGCAUGCGGUACUAU
140
AUAGUACCGCAUGCAAGGC
141
40909
AD-
941
AUGCGGUACUAUGGUAUCU
142
AGAUACCAUAGUACCGCAU
143
40915
AD-
943
GCGGUACUAUGGUAUCUGU
144
ACAGAUACCAUAGUACCGC
145
40921
AD-
956
AUCUGUGUCAAGGACAACU
146
AGUUGUCCUUGACACAGAU
147
40927
AD-
1043
CGUGAUGGGCAACUAGUGA
148
UCACUAGUUGCCCAUCACG
149
40933
AD-
1107
CCUGGGUAGAAGGUCACGA
150
UCGUGACCUUCUACCCAGG
151
40892
AD-
1158
CUCACGUGGACGCAGUAAU
152
AUUACUGCGUCCACGUGAG
153
40898
AD-
1228
GGCCAUGGUGGCGUGUUAU
154
AUAACACGCCACCAUGGCC
155
40904
AD-
1235
GUGGCGUGUUAUCCAGGCA
156
UGCCUGGAUAACACGCCAC
157
40910
AD-
1253
AAUGGGCUCGGGUACGUGA
158
UCACGUACCCGAGCCCAUU
159
40916
AD-
1261
CGGGUACGUGAGGCAUGUU
160
AACAUGCCUCACGUACCCG
161
40922
AD-
1263
GGUACGUGAGGCAUGUUGA
162
UCAACAUGCCUCACGUACC
163
40928
AD-
1272
GGCAUGUUGACAAUCCCCA
164
UGGGGAUUGUCAACAUGCC
165
40934
AD-
1305
GCAUCACCUGUAUCUAUUA
166
UAAUAGAUACAGGUGAUGC
167
40743
AD-
1329
AUCAGAACUGGGAUGUUAA
168
UUAACAUCCCAGUUCUGAU
169
40749
AD-
1335
ACUGGGAUGUUAAGGUGCA
170
UGCACCUUAACAUCCCAGU
171
40755
AD-
1399
CAACAUCGAGCCACUCUUU
172
AAAGAGUGGCUCGAUGUUG
173
40761
AD-
1534
CAGAGACAAGUAUCAGCUA
174
UAGCUGAUACUUGUCUCUG
175
40767
AD-
1537
AGACAAGUAUCAGCUAGCA
176
UGCUAGCUGAUACUUGUCU
177
40773
AD-
1555
AUCGGGACAGAAAGGUGUU
178
AACACCUUUCUGUCCCGAU
179
40779
AD-
1567
AGGUGUUCAAGUACCAGUA
180
UACUGGUACUUGAACACCU
181
40785
AD-
1708
GUGGUGUGGAGGGCACUAA
182
UUAGUGCCCUCCACACCAC
183
40744
AD-
1710
GGUGUGGAGGGCACUAAGU
184
ACUUAGUGCCCUCCACACC
185
40750
AD-
1711
GUGUGGAGGGCACUAAGUA
186
UACUUAGUGCCCUCCACAC
187
40756
AD-
1830
UGGCUGUGUCUGGUCCGUU
188
AACGGACCAGACACAGCCA
189
40762
AD-
1872
GGAUUUGGGGUUGAGGUGA
190
UCACCUCAACCCCAAAUCC
191
40768
AD-
1876
UUGGGGUUGAGGUGAGUCA
192
UGACUCACCUCAACCCCAA
193
40774
AD-
1917
GUUGGGGUGUGGGUGUCAU
194
AUGACACCCACACCCCAAC
195
40780
AD-
2038
AGGGUGCCAUGACGAGCAU
196
AUGCUCGUCAUGGCACCCU
197
40786
*Start is the 5′ most position on transcript NM_053208.4
TABLE 2D
Mouse EGNL2 Chemically modified Single Strands and Duplex
Sequences
For all the sequences in the list, ‘endolight’ chemistry was applied as
described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-40891
128
AucAGucccuucucAAGcudTsdT
198
AGCUUGAGAAGGGACUGAUdTsdT
199
AD-40897
418
GuccuuGGAGucuAGccGAdTsdT
200
UCGGCuAGACUCcAAGGACdTsdT
201
AD-40903
545
GccAcuGcuAcuAcGAccAdTsdT
202
UGGUCGuAGuAGcAGUGGCdTsdT
203
AD-40909
934
GccuuGcAuGcGGuAcuAudTsdT
204
AuAGuACCGcAUGcAAGGCdTsdT
205
AD-40915
941
AuGcGGuAcuAuGGuAucudTsdT
206
AGAuACcAuAGuACCGcAUdTsdT
207
AD-40921
943
GcGGuAcuAuGGuAucuGudTsdT
208
AcAGAuACcAuAGuACCGCdTsdT
209
AD-40927
956
AucuGuGucAAGGAcAAcudTsdT
210
AGUUGUCCUUGAcAcAGAUdTsdT
211
AD-40933
1043
cGuGAuGGGcAAcuAGuGAdTsdT
212
UcACuAGUUGCCcAUcACGdTsdT
213
AD-40892
1107
ccuGGGuAGAAGGucAcGAdTsdT
214
UCGUGACCUUCuACCcAGGdTsdT
215
AD-40898
1158
cucAcGuGGAcGcAGuAAudTsdT
216
AUuACUGCGUCcACGUGAGdTsdT
217
AD-40904
1228
GGccAuGGuGGcGuGuuAudTsdT
218
AuAAcACGCcACcAUGGCCdTsdT
219
AD-40910
1235
GuGGcGuGuuAuccAGGcAdTsdT
220
UGCCUGGAuAAcACGCcACdTsdT
221
AD-40916
1253
AAuGGGcucGGGuAcGuGAdTsdT
222
UcACGuACCCGAGCCcAUUdTsdT
223
AD-40922
1261
cGGGuAcGuGAGGcAuGuudTsdT
224
AAcAUGCCUcACGuACCCGdTsdT
225
AD-40928
1263
GGuAcGuGAGGcAuGuuGAdTsdT
226
UcAAcAUGCCUcACGuACCdTsdT
227
AD-40934
1272
GGcAuGuuGAcAAuccccAdTsdT
228
UGGGGAUUGUcAAcAUGCCdTsdT
229
AD-40743
1305
GcAucAccuGuAucuAuuAdTsdT
230
uAAuAGAuAcAGGUGAUGCdTsdT
231
AD-40749
1329
AucAGAAcuGGGAuGuuAAdTsdT
232
UuAAcAUCCcAGUUCUGAUdTsdT
233
AD-40755
1335
AcuGGGAuGuuAAGGuGcAdTsdT
234
UGcACCUuAAcAUCCcAGUdTsdT
235
AD-40761
1399
cAAcAucGAGccAcucuuudTsdT
236
AAAGAGUGGCUCGAUGUUGdTsdT
237
AD-40767
1534
cAGAGAcAAGuAucAGcuAdTsdT
238
uAGCUGAuACUUGUCUCUGdTsdT
239
AD-40773
1537
AGAcAAGuAucAGcuAGcAdTsdT
240
UGCuAGCUGAuACUUGUCUdTsdT
241
AD-40779
1555
AucGGGAcAGAAAGGuGuudTsdT
242
AAcACCUUUCUGUCCCGAUdTsdT
234
AD-40785
1567
AGGuGuucAAGuAccAGuAdTsdT
244
uACUGGuACUUGAAcACCUdTsdT
245
AD-40744
1708
GuGGuGuGGAGGGcAcuAAdTsdT
246
UuAGUGCCCUCcAcACcACdTsdT
247
AD-40750
1710
GGuGuGGAGGGcAcuAAGudTsdT
248
ACUuAGUGCCCUCcAcACCdTsdT
249
AD-40756
1711
GuGuGGAGGGcAcuAAGuAdTsdT
250
uACUuAGUGCCCUCcAcACdTsdT
251
AD-40762
1830
uGGcuGuGucuGGuccGuudTsdT
252
AACGGACcAGAcAcAGCcAdTsdT
253
AD-40768
1872
GGAuuuGGGGuuGAGGuGAdTsdT
254
UcACCUcAACCCcAAAUCCdTsdT
255
AD-40774
1876
uuGGGGuuGAGGuGAGucAdTsdT
256
UGACUcACCUcAACCCcAAdTsdT
257
AD-40780
1917
GuuGGGGuGuGGGuGucAudTsdT
258
AUGAcACCcAcACCCcAACdTsdT
259
AD-40786
2038
AGGGuGccAuGAcGAGcAudTsdT
260
AUGCUCGUcAUGGcACCCUdTsdT
261
TABLE 2E
Mouse EGNL3 Single Strands and Duplex Sequences
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-
634
CCGGCUGGGCAAAUACUAU
262
AUAGUAUUUGCCCAGCCGG
263
40745
AD-
775
GAAUUGGGACGCCAAGUUA
264
UAACUUGGCGUCCCAAUUC
265
40751
AD-
819
CGGAAGGGAAAUCGUUUGU
266
ACAAACGAUUUCCCUUCCG
267
40757
AD-
882
CAGACCGCAGGAAUCCACA
268
UGUGGAUUCCUGCGGUCUG
269
40763
AD-
922
CACCAGGUACGCUAUGACU
270
AGUCAUAGCGUACCUGGUG
271
40769
AD-
924
CCAGGUACGCUAUGACUGU
272
ACAGUCAUAGCGUACCUGG
273
40775
AD-
937
GACUGUCUGGUACUUCGAU
274
AUCGAAGUACCAGACAGUC
275
40781
AD-
1053
GGCCGCAUUCGUGUAGUAA
276
UUACUACACGAAUGCGGCC
277
40787
AD-
1055
CCGCAUUCGUGUAGUAACA
278
UGUUACUACACGAAUGCGG
279
40746
AD-
1058
CAUUCGUGUAGUAACAGUU
280
AACUGUUACUACACGAAUG
281
40752
AD-
1065
GUAGUAACAGUUCCGGAAA
282
UUUCCGGAACUGUUACUAC
283
40758
AD-
1068
GUAACAGUUCCGGAAAUGU
284
ACAUUUCCGGAACUGUUAC
285
40764
AD-
1265
CCAGCGGUUUAAAGAUAGA
286
UCUAUCUUUAAACCGCUGG
287
40770
AD-
1309
GGACUGCUUCUUAUUCGCA
288
UGCGAAUAAGAAGCAGUCC
289
40776
AD-
1312
CUGCUUCUUAUUCGCACUU
290
AAGUGCGAAUAAGAAGCAG
291
40782
AD-
1318
CUUAUUCGCACUUUAUGUA
292
UACAUAAAGUGCGAAUAAG
293
40788
AD-
1334
GUAUGCGUCCUGAUUUGAA
294
UUCAAAUCAGGACGCAUAC
295
40747
AD-
1358
GAGGUUCGCAAAGAAAUAA
296
UUAUUUCUUUGCGAACCUC
297
40753
AD-
1474
GACAGUGACGACGACCUAA
298
UUAGGUCGUCGUCACUGUC
299
40759
AD-
1480
GACGACGACCUAAUGACAU
300
AUGUCAUUAGGUCGUCGUC
301
40765
AD-
1482
CGACGACCUAAUGACAUUA
302
UAAUGUCAUUAGGUCGUCG
303
40771
AD-
1516
GCUGCUGCUUAGCAAUCGA
304
UCGAUUGCUAAGCAGCAGC
305
40777
AD-
1517
CUGCUGCUUAGCAAUCGAU
306
AUCGAUUGCUAAGCAGCAG
307
40783
AD-
1548
CACGGUGGAUGCUCCAUUU
308
AAAUGGAGCAUCCACCGUG
309
40789
AD-
1571
GGUUUACGACCCGUACUUU
310
AAAGUACGGGUCGUAAACC
311
40748
AD-
1815
CCCAACUUACAUGAUUCGU
312
ACGAAUCAUGUAAGUUGGG
313
40754
AD-
1929
GUUCAUCGUCCAUAACAAA
314
UUUGUUAUGGACGAUGAAC
315
40760
AD-
2034
CUCACUUGAGUCGUCUUGA
316
UCAAGACGACUCAAGUGAG
317
40766
AD-
2146
CCUCCCGAACUCUGUACGA
318
UCGUACAGAGUUCGGGAGG
319
40772
AD-
2157
CUGUACGAAACACCUAUUU
320
AAAUAGGUGUUUCGUACAG
321
40778
AD-
2162
CGAAACACCUAUUUUACGA
322
UCGUAAAAUAGGUGUUUCG
323
40784
AD-
2163
GAAACACCUAUUUUACGAA
324
UUCGUAAAAUAGGUGUUUC
325
40790
*Start is the 5′ most position on transcript NM_028133.2
TABLE 2F
Mouse EGNL3 Chemically modified Single Strands and Duplex
Sequences
For all the sequences in the list, ‘endolight’ chemistry was applied as
described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Start*
Sense
NO.
Antisense
NO.
AD-40745
634
ccGGcuGGGcAAAuAcuAudTsdT
326
AuAGuAUUUGCCcAGCCGGdTsdT
327
AD-40751
775
GAAuuGGGAcGccAAGuuAdTsdT
328
uAACUUGGCGUCCcAAUUCdTsdT
329
AD-40757
819
cGGAAGGGAAAucGuuuGudTsdT
330
AcAAACGAUUUCCCUUCCGdTsdT
331
AD-40763
882
cAGAccGcAGGAAuccAcAdTsdT
332
UGUGGAUUCCUGCGGUCUGdTsdT
333
AD-40769
922
cAccAGGuAcGcuAuGAcudTsdT
334
AGUcAuAGCGuACCUGGUGdTsdT
335
AD-40775
924
ccAGGuAcGcuAuGAcuGudTsdT
336
AcAGUcAuAGCGuACCUGGdTsdT
337
AD-40781
937
GAcuGucuGGuAcuucGAudTsdT
338
AUCGAAGuACcAGAcAGUCdTsdT
339
AD-40787
1053
GGccGcAuucGuGuAGuAAdTsdT
340
UuACuAcACGAAUGCGGCCdTsdT
341
AD-40746
1055
ccGcAuucGuGuAGuAAcAdTsdT
342
UGUuACuAcACGAAUGCGGdTsdT
343
AD-40752
1058
cAuucGuGuAGuAAcAGuudTsdT
344
AACUGUuACuAcACGAAUGdTsdT
345
AD-40758
1065
GuAGuAAcAGuuccGGAAAdTsdT
346
UUUCCGGAACUGUuACuACdTsdT
347
AD-40764
1068
GuAAcAGuuccGGAAAuGudTsdT
348
AcAUUUCCGGAACUGUuACdTsdT
349
AD-40770
1265
ccAGcGGuuuAAAGAuAGAdTsdT
350
UCuAUCUUuAAACCGCUGGdTsdT
351
AD-40776
1309
GGAcuGcuucuuAuucGcAdTsdT
352
UGCGAAuAAGAAGcAGUCCdTsdT
353
AD-40782
1312
cuGcuucuuAuucGcAcuudTsdT
354
AAGUGCGAAuAAGAAGcAGdTsdT
355
AD-40788
1318
cuuAuucGcAcuuuAuGuAdTsdT
356
uAcAuAAAGUGCGAAuAAGdTsdT
357
AD-40747
1334
GuAuGcGuccuGAuuuGAAdTsdT
358
UUcAAAUcAGGACGcAuACdTsdT
359
AD-40753
1358
GAGGuucGcAAAGAAAuAAdTsdT
360
UuAUUUCUUUGCGAACCUCdTsdT
361
AD-40759
1474
GAcAGuGAcGAcGAccuAAdTsdT
362
UuAGGUCGUCGUcACUGUCdTsdT
363
AD-40765
1480
GAcGAcGAccuAAuGAcAudTsdT
364
AUGUcAUuAGGUCGUCGUCdTsdT
365
AD-40771
1482
cGAcGAccuAAuGAcAuuAdTsdT
366
uAAUGUcAUuAGGUCGUCGdTsdT
367
AD-40777
1516
GcuGcuGcuuAGcAAucGAdTsdT
368
UCGAUUGCuAAGcAGcAGCdTsdT
369
AD-40783
1517
cuGcuGcuuAGcAAucGAudTsdT
370
AUCGAUUGCuAAGcAGcAGdTsdT
371
AD-40789
1548
cAcGGuGGAuGcuccAuuudTsdT
372
AAAUGGAGcAUCcACCGUGdTsdT
373
AD-40748
1571
GGuuuAcGAcccGuAcuuudTsdT
374
AAAGuACGGGUCGuAAACCdTsdT
375
AD-40754
1815
cccAAcuuAcAuGAuucGudTsdT
376
ACGAAUcAUGuAAGUUGGGdTsdT
377
AD-40760
1929
GuucAucGuccAuAAcAAAdTsdT
378
UUUGUuAUGGACGAUGAACdTsdT
379
AD-40766
2034
cucAcuuGAGucGucuuGAdTsdT
380
UcAAGACGACUcAAGUGAGdTsdT
381
AD-40772
2146
ccucccGAAcucuGuAcGAdTsdT
382
UCGuAcAGAGUUCGGGAGGdTsdT
383
AD-40778
2157
cuGuAcGAAAcAccuAuuudTsdT
384
AAAuAGGUGUUUCGuAcAGdTsdT
385
AD-40784
2162
cGAAAcAccuAuuuuAcGAdTsdT
386
UCGuAAAAuAGGUGUUUCGdTsdT
387
AD-40790
2163
GAAAcAccuAuuuuAcGAAdTsdT
388
UUCGuAAAAuAGGUGUUUCdTsdT
389
RNA Isolation, cDNA Synthesis and RT-PCR Methods
Total RNA Isolation Using MagMAX-96 Total RNA Isolation Kit (Applied Biosystem, Forer City Calif., Part #: AM1830):
Cells were harvested and lysed in 140 μl of Lysis/Binding Solution then mixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (the mixing speed was the same throughout the process). Twenty micro liters of magnetic beads and Lysis/Binding Enhancer mixture were added into cell-lysate and mixed for 5 minutes. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, magnetic beads were washed with Wash Solution 1 (isopropanol added) and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 μl Wash Solution 2 (Ethanol added), captured and supernatant was removed. 50 μl of DNase mixture (MagMax turbo DNase Buffer and Turbo DNase) was then added to the beads and they were mixed for 10 to 15 minutes. After mixing, 100 μl of RNA Rebinding Solution was added and mixed for 3 minutes. Supernatant was removed and magnetic beads were washed again with 150 μl Wash Solution 2 and mixed for 1 minute and supernatant was removed completely. The magnetic beads were mixed for 2 minutes to dry before RNA was eluted with 50 μl of water.
cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813):
A master mix of 2 μl 10× Buffer, 0.8 μl 25X dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.
Real Time PCR:
2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat # 4326317E), 0.5 μl CD274 (PD-L1) TaqMan probe (Applied Biosystems cat # Hs01125301_ml) and 5 μl Roche Probes Master Mix (Roche Cat # 04887301001) in a total of 10 μl per well in a LightCycler 480 384 well plate (Roche cat # 0472974001). Real time PCR was done in a LightCycler 480 Real Time PCR machine (Roche). Each duplex was tested in at least two independent transfections. Each transfection was assayed by qPCR in duplicate.
Real time data were analyzed using the ΔΔCt method. Each sample was normalized to GAPDH expression and knockdown was assessed relative to cells transfected with the non-targeting duplex AD-1955. IC50s were defined using a 4 parameter fit model in XLfit.
In Vitro Screening of EGLN1, EGLN2, EGLN3 siRNAs for mRNA Suppression
Mouse EGLN1 or EGLN2 or EGLN3 targeting dsRNAs (Tables 2A-F) were assayed for inhibition of endogenous EGLN1, 2, 3 expression in BNLC12 cells, using bDNA (branched DNA) assays to quantify EGLN1,2,3 mRNA. Results from single dose assays were used to select a subset of EGLN1, EGLN2 or EGLN3 dsRNA duplexes for 3 point dose response experiments to determine relative potency. The most potent siRNA for each target-EGLN1,2,3 was selected for further testing in vivo.
Cell Culture and Transfections:
The mouse liver cell line Bnlc12 (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in Dulbecco's modified Eagle's medium (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Reverse transfection was carried out by adding 5 μl of Opti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate along with 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat # 13778-150) and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotics containing 2×104 Bnlc12 cells were then added. Cells were incubated for 24 hours prior to preparation of cell lysates for branched DNA. Single dose experiments were performed at 1 nM final duplex concentration and dose response experiments were done with 1, 0.1, and 0.01 nM.
Branched DNA (bDNA) Assays-QuantiGene 2.0 (Panomics Cat #: QS0011): Used to Screen Duplexes
After a 24 hour incubation at the dose or doses stated, media was removed and cells were lysed in 100 ul Lysis buffer (Epicenter technologies and 10 μl of Proteinase-K/ml for a final concentration of 20 mg/ml) then incubated at 65° C. for 1 hour. 60 μl Working Probe Set (EGLN1, EGLN2 or EGLN3 probe for gene target and GAPDH for endogenous control) and 40 μl of cell-lysate were then added to the Capture Plates. Capture Plates were incubated at 55° C.±1° C. (approx. 16-20 hrs). The next day, the Capture Plates were washed 3 times with 1× Wash Buffer (nuclease-free water, Buffer Component 1 and Wash Buffer Component 2), then dried by centrifuging for 1 minute at 240 g. 100 μl of pre-Amplifier Working Reagent was added to the Capture Plates, which were sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C. Following a 1 hour incubation, the wash step was repeated, then 100 μl Amplifier Working Reagent was added. After 1 hour, the wash and dry steps were repeated, and 100 μl Label Probe was added. Capture plates were incubated 50° C.±1° C. for 1 hour. The plates were then washed with 1× Wash Buffer and dried, and then 100 μl Substrate was added to the Capture Plates. Capture Plates were read using the SpectraMax Luminometer (Molecular Devices, Sunnyvale, Calif.) following 5 to 15 minutes incubation. bDNA data were analyzed by (i) subtracting the average background (no lysate control) from each triplicate sample, (ii) averaging the resultant triplicate GAPDH (control probe) and EGLN1 or EGLN2 or EGLN3 (experimental probe) values, and then (iii) taking the ratio: (experimental probe-background)/(control probe-background).
Results
A summary of the single dose and 3 point dose response curve results for EGLN1, EGLN2, ELGN3-dsRNAs (siRNAs) are presented below in FIGS. 1 and 2. Single dose results are expressed as a ratio of EGLN1, or EGLN2, or EGLN3 to GAPDH mRNA in relative light units. The 3 point dose response data is expressed as % EGLN1, EGLN2 or EGLN3 mRNA relative to control untreated, assayed in BnlC12 cells.
Example 3
In Vivo Knock Down of EGLN Genes
In order to determine whether the iRNA agents to the EGLN genes were specific, knockdown studies were performed using the iRNA agents set out in Table 3.
One siRNA targeting each gene EGLN1 (AD-40894), EGLN2 (AD-40773) and EGLN3 (AD-40758) as well as a mix of all three siRNAs (AD-40894/AD-40773/AD-40758) were formulated in LNP11 (MC3) formulations to test the ability to knockdown their respective mRNAs in the liver. The experimental outline is below in Table 3 and includes control PBS group as well as a control group with an LNP11 formulation containing the Luciferase siRNA AD-1955. The individual formulations were dosed intravenously at 0.3 mg/kg into female C57B6 mice whereas the combination mix formulation was dosed at 1 mg/kg.
At 72 hours after dosing the animals were sacrificed. Plasma samples were taken and livers were removed, flash frozen then ground into powder. Small amounts (˜20 mg) of liver powder was disrupted in lysis buffer for mRNA analysis by branched DNA-QuantiGene 2.0 (Panomics cat #: QS0011). The same bDNA assay and probes used for the screening work was used. The data is expressed as percent of PBS control ratios of target (EGLN1, 2, 3) mRNA relative to GAPDH mRNA. The results are shown in FIG. 3.
It can be seen from FIG. 3 that the iRNA agents for each EGLN gene are specific to that variant. It is also evident that the mix or cocktail containing all three iRNA was effective in reducing the mRNA level of each EGLN gene.
TABLE 3
In vivo knockdown of EGLN genes
Sample
Dose
In vitro
Group
siRNA
Formulation
Size (n)
(mg/kg)
IC50
PBS
—
4
Luciferase
AD-1955
LNP11
5
0.3
(control)
EGLN1
AD-40894
LNP11
5
0.3
<10 pM
EGLN2
AD-40773
LNP11
5
0.3
~50 pM
EGLN3
AD-40758
LNP11
5
0.3
~10 pM
EGLN1, 2, 3
AD-40894
LNP11
5
1
mix
(25%)
AD-40773
(50%)
AD-40758
(25%)
Example 4
In Vivo Induction of Hepatic Erythropoietin (EPO)
In order to determine if knockdown of the three EGLN (HIF prolyl hydroxylases) genes simultaneously in the liver will induce downstream hepatic Epo (Erythropoetin) production, mice were injected IV with iRNA agents directed to each EGLN gene at 0.3 mg/kg or with a mix of all three EGLN iRNA agents (1 mg/kg) as described in Table 3 above. All iRNA agents were delivered in formulation LNP11. At 72 hours, the animals were sacrificed and livers taken for bDNA analysis. Serum was also taken for erythropoietin (EPO) measurements by ELISA kit (R&D Systems) according to the manufacturer's instructions. The results are shown in FIGS. 4A and 4B.
Only the serum samples for the PBS, Luciferase (AD-1955) and LNP11-AD-40894/AD-40773/AD-40758 (EGLN1,2,3 mix) formulation were measured for EPO. The data indicate that only serum from animals treated with the LNP11-AD-40894/AD-40773/AD-40758 treated animals showed an increase in EPO levels which was not seen in serum from animals treated with PBS or control Luciferase. Therefore, siRNA formulations that knockdown of all three EGLNs 1, 2, 3 simultaneously in liver can induce an increase in hepatic EPO production measured in serum.
Example 5
In Vivo Dose Response of EGLN in Liver
In order to evaluate the efficacy of the iRNA agents directed to EGLN genes, dose response studies were conducted for the individual EGLNs in liver. For these studies, mice (3 animals per group) were injected IV with formulations at doses outlined in Table 4. A mix of EGLN1 and EGLN3 formulations were tested to confirm if co-injection of individual LNP11 formulations with siRNA against single targets worked as well as injection of a single formulation with siRNAs against all 3 EGLN targets. At 72 hours, the animals were sacrificed and livers taken for bDNA and serum taken for Epo measurements by ELISA. The results are shown in FIG. 5.
Results
It was found that all three formulations LNP11-40894, LNP11-40773, and LNP11-40758 dose dependently knocked down the respective mRNA levels of EGLN1, EGLN2 and EGLN3 after IV administration into C57B6 mice. The relative IC50 values in vivo were less than 0.033 for LNP11-40894 targeting EGLN1, less than 0.033 for LNP11-40773 targeting of EGLN2 and approximately 0.05 for LNP11-40758. Furthermore, it was possible to detect knockdown of EGLN1 and EGLN3 mRNAs by injection of LNP11-40894 and LNP11-40758, suggesting that the siRNAs don't have to be inside the same liposome together to silence both targets simultaneously.
TABLE 4
In vivo knockdown of EGLN genes
Sample
Dose
In vitro
Group
siRNA
Formulation
Size (n)
(mg/kg)
IC50
PBS
—
3
Luciferase
AD-1955
LNP11
3
1
EGLN1
AD-40894
LNP11
3 (12
1
<10 pM
total)
0.33
0.1
0.033
EGLN2
AD-40773
LNP11
3 (12
1
~50 pM
total)
0.33
0.1
0.033
EGLN3
AD-40758
LNP11
3 (12
1
~10 pM
total)
0.33
0.1
0.033
EGLN1, 3
AD-40894
LNP11
3
0.67/
mix
(67%)
0.33
AD-40758
(33%)
Example 6
In Vivo Production of Erythropoietin and Hematology
In order to determine whether administration of an EGLN iRNA cocktail was capable of increasing erythropoietin expression in vivo, a study was designed according to Table 5. Female C57B6 mice were dosed IV with PBS or LNP11-1955 luciferase controls or two different mixes of EGLN siRNA formulations at two different doses 1.5 or 1.33 mg/kg respectively. On day 5 after the first dose plasma samples were taken from each animal for hematology measurements. On day 7, a second dose of the same amount of a mix of LNP11 formulations or controls was given. Then on day 10 a second set of plasma samples were taken, animals were sacrificed and livers were harvested for measurement of EGLN1, EGLN2, EGLN3 and EPO mRNA measurements again by branched DNA analysis. At 72 hours, after the 1st dose blood was drawn for hematology measurements including a count of reticulocytes, red blood cells, hemoglobin measurements and hematocrit levels. At 72 hours after the 2nd dose animals were sacrificed and livers taken for bDNA analysis. The Week 1 data are shown in FIGS. 6 and 7 while Week 2 data are shown in FIGS. 8 and 9.
TABLE 5
In vivo knockdown of EGLN genes
Sample
Dose
Group
siRNA
Formulation
size (n)
(mg/kg)
PBS
—
5
Luciferase
AD-1955
LNP11
5
1
EGLN1, 2, 3
AD-40894 (.375 mpk)
LNP11
15
1.5
mix 1
AD-40773 (.75 mpk)
AD-40758 (.375 mpk)
EGLN1, 2, 3
AD-40894 (.25 mpk)
LNP11
10
1.33
mix 2
AD-40773 (.5 mpk)
AD-40758 (.58 mpk)
It can be seen from FIGS. 6-9 that in both Weeks 1 and 2 that both mix 1 and mix 2 result in observable changes. It was found that by day 5 after the first dose a large increase in reticulocyte levels and a small increase in hematocrit readouts could be detected. By day 10, now after 2 injections of the mix of LNP11 formulations with EGLN1, EGLN2 and EGLN3 siRNAs, a considerable increase in reticulocytes versus control was observed with an even larger increase in hematocrit, RBC count and hemoglobin levels in the plasma. Collectively, knockdown of EGLN1,2,3 led to an increase in liver EPO mRNA and stimulated erythropiesis.
Furthermore, it was found that injection of the mix of 3 LNPs targeting each EGLN gene resulted in knockdown of all three EGLN targets EGLN1, ELGN2, and EGLN3 while simultaneously leading to an increase of EPO mRNA after 2 doses at day 10. The data are shown in FIG. 10. The luciferase siRNA and PBS treated animals had EPO mRNA levels at essentially background levels in the liver whereas in the EGLN siRNA mix treated group there was strong EPO mRNA expression. EGLN1, EGLN2, EGLN3, and EPO mRNA levels were normalized to housekeeping GAPDH control and data is expressed as a percentage of the PBS control expression.
From these data, it may be concluded that simultaneous knockdown of all three EGLN genes in the liver is possible with each siRNA in their own LNP formulations, then mixing them prior to injection. The knockdown of the 3 EGLN genes lead to a very dramatic increased expression of EPO mRNA as compared to the PBS control or Luciferase siRNA treated groups where liver EPO mRNA was undetectable and at background levels of the assay. Furthermore, it was found that by turning on EPO mRNA expression in the liver by knocking down the 3 EGLN genes a dramatic increase in erythropoiesis occurs. This could be measured in the blood from dosed animals where a dramatic increase in reticulocytes or (immature red blood cells) was observed even after the first dose of EGLN1,2,3 siRNA mix treatment. After the second dose it was evident that a significant increase in not only reticulocytes but also RBC count, hemoglobin and Hematocrit measurements was occurring.
Example 7
Design of siRNA Targeting Human EGLN Genes
Oligonucleotide design was carried out to identify siRNAs targeting the genes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. The design process used the EGLN transcript NM—022051.2 for EGLN1 (SEQ ID NO: 390), NM—053046.2 for EGLN2 (SEQ ID NO: 391), and NM—022073.3 for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBI Refseq collection. Start refers to the 5′ most position on the target.
It should be understood that while the sequences disclosed in Tables 6A-C are represented as 19mer oligonucleotides, the duplexes formed from such oligonucleotides may be 19mer blunt ended constructs or may be modified by the addition of one or more nucleotides on the 3′ end of the strands, preferably a dTdT modification to produce 21mer duplexes having 2 nucleotide 3′ overhangs.
TABLE 6A
Human EGNL1 Single Strands and Duplex Sequences
SEQ
SEQ
ID
ID
Start
Sense Sequence (5′ to 3′)
NO.
Antisense Sequence (5′ to 3′)
NO.
40
AGAGACACAAGGCUUUGUU
393
AACAAAGCCUUGUGUCUCU
394
55
UGUUUGCCCCAGAGUAUUA
395
UAAUACUCUGGGGCAAACA
396
59
UGCCCCAGAGUAUUAGUUA
397
UAACUAAUACUCUGGGGCA
398
60
GCCCCAGAGUAUUAGUUAA
399
UUAACUAAUACUCUGGGGC
400
64
CAGAGUAUUAGUUAACCCA
401
UGGGUUAACUAAUACUCUG
402
70
AUUAGUUAACCCACCUAGU
403
ACUAGGUGGGUUAACUAAU
404
73
AGUUAACCCACCUAGUGCU
405
AGCACUAGGUGGGUUAACU
406
77
AACCCACCUAGUGCUCCUA
407
UAGGAGCACUAGGUGGGUU
408
79
CCCACCUAGUGCUCCUAAU
409
AUUAGGAGCACUAGGUGGG
410
86
AGUGCUCCUAAUCAUACAA
411
UUGUAUGAUUAGGAGCACU
412
132
GCCUCACUCUCUAUUUGUU
413
AACAAAUAGAGAGUGAGGC
414
153
ACCUUCUGUAAAAUUGGUA
415
UACCAAUUUUACAGAAGGU
416
168
GGUAGAAUAAUAGUACCCA
417
UGGGUACUAUUAUUCUACC
418
170
UAGAAUAAUAGUACCCACU
419
AGUGGGUACUAUUAUUCUA
420
171
AGAAUAAUAGUACCCACUU
421
AAGUGGGUACUAUUAUUCU
422
179
AGUACCCACUUCAUAGCAU
423
AUGCUAUGAAGUGGGUACU
424
201
AUGAUGAUUAAAUUGGUUA
425
UAACCAAUUUAAUCAUCAU
426
235
UUAGAACACAGAUUGGGCA
427
UGCCCAAUCUGUGUUCUAA
428
245
GAUUGGGCACAUAACAGCA
429
UGCUGUUAUGUGCCCAAUC
430
249
GGGCACAUAACAGCAAGCA
431
UGCUUGCUGUUAUGUGCCC
432
255
AUAACAGCAAGCACCACAU
433
AUGUGGUGCUUGCUGUUAU
434
287
AAAUUCCUUUGUGUUGCCU
435
AGGCAACACAAAGGAAUUU
436
292
CCUUUGUGUUGCCUUCCGU
437
ACGGAAGGCAACACAAAGG
438
293
CUUUGUGUUGCCUUCCGUU
439
AACGGAAGGCAACACAAAG
440
295
UUGUGUUGCCUUCCGUUAA
441
UUAACGGAAGGCAACACAA
442
296
UGUGUUGCCUUCCGUUAAA
443
UUUAACGGAAGGCAACACA
444
298
UGUUGCCUUCCGUUAAAGU
445
ACUUUAACGGAAGGCAACA
446
299
GUUGCCUUCCGUUAAAGUU
447
AACUUUAACGGAAGGCAAC
448
336
AAUAAAUACUUGCAUGACA
449
UGUCAUGCAAGUAUUUAUU
450
360
AAGUCUCUCUAUAACAUCU
451
AGAUGUUAUAGAGAGACUU
452
368
CUAUAACAUCUGAGUAAGU
453
ACUUACUCAGAUGUUAUAG
454
375
AUCUGAGUAAGUGGCGGCU
455
AGCCGCCACUUACUCAGAU
456
389
CGGCUGCGACAAUGCUACU
457
AGUAGCAUUGUCGCAGCCG
458
394
GCGACAAUGCUACUGGAGU
459
ACUCCAGUAGCAUUGUCGC
460
395
CGACAAUGCUACUGGAGUU
461
AACUCCAGUAGCAUUGUCG
462
411
GUUCCAGAAUCGUGUUGGU
463
ACCAACACGAUUCUGGAAC
464
428
GUGACAAGAUUGUUCACCA
465
UGGUGAACAAUCUUGUCAC
466
434
AGAUUGUUCACCAGCAUAU
467
AUAUGCUGGUGAACAAUCU
468
439
GUUCACCAGCAUAUGGUGU
469
ACACCAUAUGCUGGUGAAC
470
444
CCAGCAUAUGGUGUGGUGA
471
UCACCACACCAUAUGCUGG
472
453
GGUGUGGUGAAAACUCACU
473
AGUGAGUUUUCACCACACC
474
455
UGUGGUGAAAACUCACUAA
475
UUAGUGAGUUUUCACCACA
476
457
UGGUGAAAACUCACUAAUU
477
AAUUAGUGAGUUUUCACCA
478
458
GGUGAAAACUCACUAAUUU
479
AAAUUAGUGAGUUUUCACC
480
488
AGAUUAUUAAGCCUGAAUA
481
UAUUCAGGCUUAAUAAUCU
482
491
UUAUUAAGCCUGAAUAGGU
483
ACCUAUUCAGGCUUAAUAA
484
493
AUUAAGCCUGAAUAGGUGA
485
UCACCUAUUCAGGCUUAAU
486
494
UUAAGCCUGAAUAGGUGAA
487
UUCACCUAUUCAGGCUUAA
488
495
UAAGCCUGAAUAGGUGAAA
489
UUUCACCUAUUCAGGCUUA
490
519
GAAAUCAAGGAUCUUUGGA
491
UCCAAAGAUCCUUGAUUUC
492
579
UUAAAGUGUUGCAAGUGUU
493
AACACUUGCAACACUUUAA
494
597
UCUAUUUGAUGGAUUAAGU
495
ACUUAAUCCAUCAAAUAGA
496
598
CUAUUUGAUGGAUUAAGUA
497
UACUUAAUCCAUCAAAUAG
498
599
UAUUUGAUGGAUUAAGUAU
499
AUACUUAAUCCAUCAAAUA
500
600
AUUUGAUGGAUUAAGUAUA
501
UAUACUUAAUCCAUCAAAU
502
601
UUUGAUGGAUUAAGUAUAU
503
AUAUACUUAAUCCAUCAAA
504
610
UUAAGUAUAUUUAGGAUAU
505
AUAUCCUAAAUAUACUUAA
506
611
UAAGUAUAUUUAGGAUAUA
507
UAUAUCCUAAAUAUACUUA
508
687
UGAUAUGGACAUCUAUUCU
509
AGAAUAGAUGUCCAUAUCA
510
688
GAUAUGGACAUCUAUUCUU
511
AAGAAUAGAUGUCCAUAUC
512
706
UUUAAGUAAACUUCAAUGA
513
UCAUUGAAGUUUACUUAAA
514
721
AUGAAAAUAUAUGAGUAGA
515
UCUACUCAUAUAUUUUCAU
516
724
AAAAUAUAUGAGUAGAGCA
517
UGCUCUACUCAUAUAUUUU
518
725
AAAUAUAUGAGUAGAGCAU
519
AUGCUCUACUCAUAUAUUU
520
726
AAUAUAUGAGUAGAGCAUA
521
UAUGCUCUACUCAUAUAUU
522
727
AUAUAUGAGUAGAGCAUAU
523
AUAUGCUCUACUCAUAUAU
524
728
UAUAUGAGUAGAGCAUAUA
525
UAUAUGCUCUACUCAUAUA
526
730
UAUGAGUAGAGCAUAUAGA
527
UCUAUAUGCUCUACUCAUA
528
771
ACCACAGACUGAAAUAGCA
529
UGCUAUUUCAGUCUGUGGU
530
827
GGAAUGAGUCCUCCUAGUA
531
UACUAGGAGGACUCAUUCC
532
828
GAAUGAGUCCUCCUAGUAA
533
UUACUAGGAGGACUCAUUC
534
829
AAUGAGUCCUCCUAGUAAA
535
UUUACUAGGAGGACUCAUU
536
832
GAGUCCUCCUAGUAAAGUU
537
AACUUUACUAGGAGGACUC
538
849
UUCCUGCUCUUGUGAAUAA
539
UUAUUCACAAGAGCAGGAA
540
859
UGUGAAUAAUUAAGCCUCA
541
UGAGGCUUAAUUAUUCACA
542
868
UUAAGCCUCAUGUAUAAUU
543
AAUUAUACAUGAGGCUUAA
544
872
GCCUCAUGUAUAAUUACUA
545
UAGUAAUUAUACAUGAGGC
546
901
AAGCCUAAGAAGUAUUAGA
547
UCUAAUACUUCUUAGGCUU
548
903
GCCUAAGAAGUAUUAGACU
549
AGUCUAAUACUUCUUAGGC
550
973
UUAAAUGCUUAUUUUCGUA
551
UACGAAAAUAAGCAUUUAA
552
978
UGCUUAUUUUCGUAAGCCA
553
UGGCUUACGAAAAUAAGCA
554
984
UUUUCGUAAGCCAUGAGAU
555
AUCUCAUGGCUUACGAAAA
556
996
AUGAGAUAGCUCCUUUAUA
557
UAUAAAGGAGCUAUCUCAU
558
1042
UGGAUUUUAUUAGUGCAAA
559
UUUGCACUAAUAAAAUCCA
560
1062
GGCAGAGCUAGCAAUUCCU
561
AGGAAUUGCUAGCUCUGCC
562
1105
AUUCAUCCCUCUUUUAGGA
563
UCCUAAAAGAGGGAUGAAU
564
1159
UGCCUCCUGCAUUGGACUA
565
UAGUCCAAUGCAGGAGGCA
566
1160
GCCUCCUGCAUUGGACUAU
567
AUAGUCCAAUGCAGGAGGC
568
1162
CUCCUGCAUUGGACUAUGU
569
ACAUAGUCCAAUGCAGGAG
570
1179
GUGUCUCUGAGUGUAGUAU
571
AUACUACACUCAGAGACAC
572
1185
CUGAGUGUAGUAUGACUAA
573
UUAGUCAUACUACACUCAG
574
1186
UGAGUGUAGUAUGACUAAU
575
AUUAGUCAUACUACACUCA
576
1187
GAGUGUAGUAUGACUAAUU
577
AAUUAGUCAUACUACACUC
578
1189
GUGUAGUAUGACUAAUUCA
579
UGAAUUAGUCAUACUACAC
580
1211
GUUUGUCAAGGACUCUCAA
581
UUGAGAGUCCUUGACAAAC
582
1216
UCAAGGACUCUCAAUGCAU
583
AUGCAUUGAGAGUCCUUGA
584
1221
GACUCUCAAUGCAUUUGUU
585
AACAAAUGCAUUGAGAGUC
586
1233
AUUUGUUGAACAGCCUAAU
587
AUUAGGCUGUUCAACAAAU
588
1237
GUUGAACAGCCUAAUUAGU
589
ACUAAUUAGGCUGUUCAAC
590
1238
UUGAACAGCCUAAUUAGUA
591
UACUAAUUAGGCUGUUCAA
592
1242
ACAGCCUAAUUAGUAAUGU
593
ACAUUACUAAUUAGGCUGU
594
1244
AGCCUAAUUAGUAAUGUCU
595
AGACAUUACUAAUUAGGCU
596
1254
GUAAUGUCUGCAACAAUGA
597
UCAUUGUUGCAGACAUUAC
598
1285
UUUAAUAAAGCUCUGGGAA
599
UUCCCAGAGCUUUAUUAAA
600
1286
UUAAUAAAGCUCUGGGAAA
601
UUUCCCAGAGCUUUAUUAA
602
1293
AGCUCUGGGAAAGUAGGAU
603
AUCCUACUUUCCCAGAGCU
604
1296
UCUGGGAAAGUAGGAUACA
605
UGUAUCCUACUUUCCCAGA
606
1303
AAGUAGGAUACACAUAAGA
607
UCUUAUGUGUAUCCUACUU
608
1308
GGAUACACAUAAGACAGGU
609
ACCUGUCUUAUGUGUAUCC
610
1314
ACAUAAGACAGGUCUAGGU
611
ACCUAGACCUGUCUUAUGU
612
1319
AGACAGGUCUAGGUCUAAA
613
UUUAGACCUAGACCUGUCU
614
1320
GACAGGUCUAGGUCUAAAU
615
AUUUAGACCUAGACCUGUC
616
1323
AGGUCUAGGUCUAAAUUCU
617
AGAAUUUAGACCUAGACCU
618
1324
GGUCUAGGUCUAAAUUCUU
619
AAGAAUUUAGACCUAGACC
620
1328
UAGGUCUAAAUUCUUUACA
621
UGUAAAGAAUUUAGACCUA
622
1338
UUCUUUACAGAAACUUGGA
623
UCCAAGUUUCUGUAAAGAA
624
1403
GUUUCCCAAAGGACAAGCU
625
AGCUUGUCCUUUGGGAAAC
626
1434
CAUCCUCUUUCACUUGAUU
627
AAUCAAGUGAAAGAGGAUG
628
1470
UUUACGCAUGCAGCAGGAU
629
AUCCUGCUGCAUGCGUAAA
630
1471
UUACGCAUGCAGCAGGAUU
631
AAUCCUGCUGCAUGCGUAA
632
1482
GCAGGAUUUUAUAACAGUU
633
AACUGUUAUAAAAUCCUGC
634
1572
UGGUUUACAAUAAUUCCUU
635
AAGGAAUUAUUGUAAACCA
636
1606
AAUACAUAUUACAACUUUU
637
AAAAGUUGUAAUAUGUAUU
638
1625
UAAGUUUGGAAGGCUAUAU
639
AUAUAGCCUUCCAAACUUA
640
1626
AAGUUUGGAAGGCUAUAUU
641
AAUAUAGCCUUCCAAACUU
642
1629
UUUGGAAGGCUAUAUUUCA
643
UGAAAUAUAGCCUUCCAAA
644
1651
ACUGAAGUUACAGUAUACU
645
AGUAUACUGUAACUUCAGU
646
1653
UGAAGUUACAGUAUACUCA
647
UGAGUAUACUGUAACUUCA
648
1654
GAAGUUACAGUAUACUCAA
649
UUGAGUAUACUGUAACUUC
650
1665
AUACUCAAGUGAUACACAA
651
UUGUGUAUCACUUGAGUAU
652
1673
GUGAUACACAAGCCUAGCA
653
UGCUAGGCUUGUGUAUCAC
654
1678
ACACAAGCCUAGCACCCCA
655
UGGGGUGCUAGGCUUGUGU
656
1693
CCCACUUUCCACAUAGUGU
657
ACACUAUGUGGAAAGUGGG
658
1697
CUUUCCACAUAGUGUUCGA
659
UCGAACACUAUGUGGAAAG
660
1698
UUUCCACAUAGUGUUCGAU
661
AUCGAACACUAUGUGGAAA
662
1699
UUCCACAUAGUGUUCGAUA
663
UAUCGAACACUAUGUGGAA
664
1700
UCCACAUAGUGUUCGAUAA
665
UUAUCGAACACUAUGUGGA
666
1701
CCACAUAGUGUUCGAUAAA
667
UUUAUCGAACACUAUGUGG
668
1705
AUAGUGUUCGAUAAAGAUU
669
AAUCUUUAUCGAACACUAU
670
1709
UGUUCGAUAAAGAUUGAUA
671
UAUCAAUCUUUAUCGAACA
672
1711
UUCGAUAAAGAUUGAUAAA
673
UUUAUCAAUCUUUAUCGAA
674
1721
AUUGAUAAACUCGAAAUCA
675
UGAUUUCGAGUUUAUCAAU
676
1723
UGAUAAACUCGAAAUCACA
677
UGUGAUUUCGAGUUUAUCA
678
1725
AUAAACUCGAAAUCACAGA
679
UCUGUGAUUUCGAGUUUAU
680
1729
ACUCGAAAUCACAGACCUU
681
AAGGUCUGUGAUUUCGAGU
682
1740
CAGACCUUUUAAUUCUUAA
683
UUAAGAAUUAAAAGGUCUG
684
1788
GGCUUAUUUCUGGUAAGGU
685
ACCUUACCAGAAAUAAGCC
686
1790
CUUAUUUCUGGUAAGGUUU
687
AAACCUUACCAGAAAUAAG
688
1829
AAUUGUAUUCAUCCGCGCA
689
UGCGCGGAUGAAUACAAUU
690
1832
UGUAUUCAUCCGCGCAGCA
691
UGCUGCGCGGAUGAAUACA
692
1834
UAUUCAUCCGCGCAGCACA
693
UGUGCUGCGCGGAUGAAUA
694
1864
AAAUAAAUGUGAGAGUCGU
695
ACGACUCUCACAUUUAUUU
696
1866
AUAAAUGUGAGAGUCGUUA
697
UAACGACUCUCACAUUUAU
698
1867
UAAAUGUGAGAGUCGUUAA
699
UUAACGACUCUCACAUUUA
700
1870
AUGUGAGAGUCGUUAAUGU
701
ACAUUAACGACUCUCACAU
702
1873
UGAGAGUCGUUAAUGUAGU
703
ACUACAUUAACGACUCUCA
704
1874
GAGAGUCGUUAAUGUAGUA
705
UACUACAUUAACGACUCUC
706
1876
GAGUCGUUAAUGUAGUACU
707
AGUACUACAUUAACGACUC
708
1884
AAUGUAGUACUGCUCAUUU
709
AAAUGAGCAGUACUACAUU
710
1917
CUUUUCAGGAAUAAUCCCA
711
UGGGAUUAUUCCUGAAAAG
712
1963
CAUUGAUUACAUUUAACUU
713
AAGUUAAAUGUAAUCAAUG
714
1966
UGAUUACAUUUAACUUGGU
715
ACCAAGUUAAAUGUAAUCA
716
1972
CAUUUAACUUGGUAGCCCA
717
UGGGCUACCAAGUUAAAUG
718
1974
UUUAACUUGGUAGCCCAAA
719
UUUGGGCUACCAAGUUAAA
720
1978
ACUUGGUAGCCCAAAAUUU
721
AAAUUUUGGGCUACCAAGU
722
1981
UGGUAGCCCAAAAUUUCUU
723
AAGAAAUUUUGGGCUACCA
724
1990
AAAAUUUCUUCAUGGGGUU
725
AACCCCAUGAAGAAAUUUU
726
2005
GGUUUUGAACUCGGCGGGA
727
UCCCGCCGAGUUCAAAACC
728
2006
GUUUUGAACUCGGCGGGAU
729
AUCCCGCCGAGUUCAAAAC
730
2007
UUUUGAACUCGGCGGGAUU
731
AAUCCCGCCGAGUUCAAAA
732
2008
UUUGAACUCGGCGGGAUUU
733
AAAUCCCGCCGAGUUCAAA
734
2012
AACUCGGCGGGAUUUCAAA
735
UUUGAAAUCCCGCCGAGUU
736
2079
UACCUUUAAACUAGGUCGA
737
UCGACCUAGUUUAAAGGUA
738
2081
CCUUUAAACUAGGUCGAAA
739
UUUCGACCUAGUUUAAAGG
740
2090
UAGGUCGAAACGGGGCGCA
741
UGCGCCCCGUUUCGACCUA
742
2091
AGGUCGAAACGGGGCGCAA
743
UUGCGCCCCGUUUCGACCU
744
2093
GUCGAAACGGGGCGCAAGA
745
UCUUGCGCCCCGUUUCGAC
746
2097
AAACGGGGCGCAAGAGAUU
747
AAUCUCUUGCGCCCCGUUU
748
2102
GGGCGCAAGAGAUUGGAUU
749
AAUCCAAUCUCUUGCGCCC
750
2103
GGCGCAAGAGAUUGGAUUA
751
UAAUCCAAUCUCUUGCGCC
752
2104
GCGCAAGAGAUUGGAUUAA
753
UUAAUCCAAUCUCUUGCGC
754
2106
GCAAGAGAUUGGAUUAACA
755
UGUUAAUCCAAUCUCUUGC
756
2109
AGAGAUUGGAUUAACACCA
757
UGGUGUUAAUCCAAUCUCU
758
2113
AUUGGAUUAACACCAUAGU
759
ACUAUGGUGUUAAUCCAAU
760
2122
ACACCAUAGUAAUACUUAU
761
AUAAGUAUUACUAUGGUGU
762
2123
CACCAUAGUAAUACUUAUU
763
AAUAAGUAUUACUAUGGUG
764
2130
GUAAUACUUAUUUUGUUCU
765
AGAACAAAAUAAGUAUUAC
766
2158
CAGGGCUUCUUGAAAUAGA
767
UCUAUUUCAAGAAGCCCUG
768
2171
AAUAGAGGCUGUAUGGUGU
769
ACACCAUACAGCCUCUAUU
770
2172
AUAGAGGCUGUAUGGUGUA
771
UACACCAUACAGCCUCUAU
772
2179
CUGUAUGGUGUAAUGGAAA
773
UUUCCAUUACACCAUACAG
774
2233
UUCAGUCCCAGUUUUGCGU
775
ACGCAAAACUGGGACUGAA
776
2235
CAGUCCCAGUUUUGCGUGA
777
UCACGCAAAACUGGGACUG
778
2239
CCCAGUUUUGCGUGACCUU
779
AAGGUCACGCAAAACUGGG
780
2298
CUGCAAAAUGAGGAUCGCA
781
UGCGAUCCUCAUUUUGCAG
782
2305
AUGAGGAUCGCAAUAGCCA
783
UGGCUAUUGCGAUCCUCAU
784
2308
AGGAUCGCAAUAGCCACCU
785
AGGUGGCUAUUGCGAUCCU
786
2309
GGAUCGCAAUAGCCACCUU
787
AAGGUGGCUAUUGCGAUCC
788
2316
AAUAGCCACCUUGCAACCU
789
AGGUUGCAAGGUGGCUAUU
790
2321
CCACCUUGCAACCUUGACU
791
AGUCAAGGUUGCAAGGUGG
792
2328
GCAACCUUGACUGGAGCGA
793
UCGCUCCAGUCAAGGUUGC
794
2338
CUGGAGCGAGCCUCGCACA
795
UGUGCGAGGCUCGCUCCAG
796
2382
AGCCAUGAUUACGCCGCCU
797
AGGCGGCGUAAUCAUGGCU
798
2383
GCCAUGAUUACGCCGCCUU
799
AAGGCGGCGUAAUCAUGGC
800
2435
UCCAGCAGGUGUAGGCGCU
801
AGCGCCUACACCUGCUGGA
802
2573
AGGGAAAGCGGGCGACCCA
803
UGGGUCGCCCGCUUUCCCU
804
2576
GAAAGCGGGCGACCCACCU
805
AGGUGGGUCGCCCGCUUUC
806
2761
GAGCGAGUGGCGCCCGUAU
807
AUACGGGCGCCACUCGCUC
808
2766
AGUGGCGCCCGUAUGCCCU
809
AGGGCAUACGGGCGCCACU
810
2885
CAGGUUGCCAUUCGCCGCA
811
UGCGGCGAAUGGCAACCUG
812
2887
GGUUGCCAUUCGCCGCACA
813
UGUGCGGCGAAUGGCAACC
814
2895
UUCGCCGCACAGGCCCUAU
815
AUAGGGCCUGUGCGGCGAA
816
2896
UCGCCGCACAGGCCCUAUU
817
AAUAGGGCCUGUGCGGCGA
818
3033
GCGGGUGCAUGGCGCAGUA
819
UACUGCGCCAUGCACCCGC
820
3034
CGGGUGCAUGGCGCAGUAA
821
UUACUGCGCCAUGCACCCG
822
3042
UGGCGCAGUAACGGCCCCU
823
AGGGGCCGUUACUGCGCCA
824
3043
GGCGCAGUAACGGCCCCUA
825
UAGGGGCCGUUACUGCGCC
826
3473
ACGCGGCCAAGGGAAAAGU
827
ACUUUUCCCUUGGCCGCGU
828
3608
CCCGCUCAUCGCUGUUCCA
829
UGGAACAGCGAUGAGCGGG
830
3626
AGGAGAAGGCGAACCUGUA
831
UACAGGUUCGCCUUCUCCU
832
3650
CAAGCAACACGCCCGGGGA
833
UCCCCGGGCGUGUUGCUUG
834
3695
GGCCCAACGGGCAGACGAA
835
UUCGUCUGCCCGUUGGGCC
836
3731
AGCUGGCGCUCGAGUACAU
837
AUGUACUCGAGCGCCAGCU
838
3734
UGGCGCUCGAGUACAUCGU
839
ACGAUGUACUCGAGCGCCA
840
3739
CUCGAGUACAUCGUGCCGU
841
ACGGCACGAUGUACUCGAG
842
3745
UACAUCGUGCCGUGCAUGA
843
UCAUGCACGGCACGAUGUA
844
3748
AUCGUGCCGUGCAUGAACA
845
UGUUCAUGCACGGCACGAU
846
3752
UGCCGUGCAUGAACAAGCA
847
UGCUUGUUCAUGCACGGCA
848
3762
GAACAAGCACGGCAUCUGU
849
ACAGAUGCCGUGCUUGUUC
850
3797
UCGGCAAGGAGACCGGACA
851
UGUCCGGUCUCCUUGCCGA
852
3809
CCGGACAGCAGAUCGGCGA
853
UCGCCGAUCUGCUGUCCGG
854
3842
UGCACGACACCGGGAAGUU
855
AACUUCCCGGUGUCGUGCA
856
3854
GGAAGUUCACGGACGGGCA
857
UGCCCGUCCGUGAACUUCC
858
3901
AAGGACAUCCGAGGCGAUA
859
UAUCGCCUCGGAUGUCCUU
860
3902
AGGACAUCCGAGGCGAUAA
861
UUAUCGCCUCGGAUGUCCU
862
3904
GACAUCCGAGGCGAUAAGA
863
UCUUAUCGCCUCGGAUGUC
864
3905
ACAUCCGAGGCGAUAAGAU
865
AUCUUAUCGCCUCGGAUGU
866
3907
AUCCGAGGCGAUAAGAUCA
867
UGAUCUUAUCGCCUCGGAU
868
3913
GGCGAUAAGAUCACCUGGA
869
UCCAGGUGAUCUUAUCGCC
870
3917
AUAAGAUCACCUGGAUCGA
871
UCGAUCCAGGUGAUCUUAU
872
3922
AUCACCUGGAUCGAGGGCA
873
UGCCCUCGAUCCAGGUGAU
874
3939
CAAGGAGCCCGGCUGCGAA
875
UUCGCAGCCGGGCUCCUUG
876
3943
GAGCCCGGCUGCGAAACCA
877
UGGUUUCGCAGCCGGGCUC
878
3944
AGCCCGGCUGCGAAACCAU
879
AUGGUUUCGCAGCCGGGCU
880
3950
GCUGCGAAACCAUUGGGCU
881
AGCCCAAUGGUUUCGCAGC
882
3953
GCGAAACCAUUGGGCUGCU
883
AGCAGCCCAAUGGUUUCGC
884
3978
CAGCAUGGACGACCUGAUA
885
UAUCAGGUCGUCCAUGCUG
886
3983
UGGACGACCUGAUACGCCA
887
UGGCGUAUCAGGUCGUCCA
888
3987
CGACCUGAUACGCCACUGU
889
ACAGUGGCGUAUCAGGUCG
890
3988
GACCUGAUACGCCACUGUA
891
UACAGUGGCGUAUCAGGUC
892
3994
AUACGCCACUGUAACGGGA
893
UCCCGUUACAGUGGCGUAU
894
4024
UACAAAAUCAAUGGCCGGA
895
UCCGGCCAUUGAUUUUGUA
896
4028
AAAUCAAUGGCCGGACGAA
897
UUCGUCCGGCCAUUGAUUU
898
4029
AAUCAAUGGCCGGACGAAA
899
UUUCGUCCGGCCAUUGAUU
900
4033
AAUGGCCGGACGAAAGCCA
901
UGGCUUUCGUCCGGCCAUU
902
4037
GCCGGACGAAAGCCAUGGU
903
ACCAUGGCUUUCGUCCGGC
904
4038
CCGGACGAAAGCCAUGGUU
905
AACCAUGGCUUUCGUCCGG
906
4047
AGCCAUGGUUGCUUGUUAU
907
AUAACAAGCAACCAUGGCU
908
4054
GUUGCUUGUUAUCCGGGCA
909
UGCCCGGAUAACAAGCAAC
910
4055
UUGCUUGUUAUCCGGGCAA
911
UUGCCCGGAUAACAAGCAA
912
4066
CCGGGCAAUGGAACGGGUU
913
AACCCGUUCCAUUGCCCGG
914
4067
CGGGCAAUGGAACGGGUUA
915
UAACCCGUUCCAUUGCCCG
916
4068
GGGCAAUGGAACGGGUUAU
917
AUAACCCGUUCCAUUGCCC
918
4070
GCAAUGGAACGGGUUAUGU
919
ACAUAACCCGUUCCAUUGC
920
4076
GAACGGGUUAUGUACGUCA
921
UGACGUACAUAACCCGUUC
922
4077
AACGGGUUAUGUACGUCAU
923
AUGACGUACAUAACCCGUU
924
4079
CGGGUUAUGUACGUCAUGU
925
ACAUGACGUACAUAACCCG
926
4080
GGGUUAUGUACGUCAUGUU
927
AACAUGACGUACAUAACCC
928
4082
GUUAUGUACGUCAUGUUGA
929
UCAACAUGACGUACAUAAC
930
4084
UAUGUACGUCAUGUUGAUA
931
UAUCAACAUGACGUACAUA
932
4085
AUGUACGUCAUGUUGAUAA
933
UUAUCAACAUGACGUACAU
934
4089
ACGUCAUGUUGAUAAUCCA
935
UGGAUUAUCAACAUGACGU
936
4090
CGUCAUGUUGAUAAUCCAA
937
UUGGAUUAUCAACAUGACG
938
4113
AGAUGGAAGAUGUGUGACA
939
UGUCACACAUCUUCCAUCU
940
4127
UGACAUGUAUAUAUUAUCU
941
AGAUAAUAUAUACAUGUCA
942
4153
GACUGGGAUGCCAAGGUAA
943
UUACCUUGGCAUCCCAGUC
944
4163
CCAAGGUAAGUGGAGGUAU
945
AUACCUCCACUUACCUUGG
946
4172
GUGGAGGUAUACUUCGAAU
947
AUUCGAAGUAUACCUCCAC
948
4173
UGGAGGUAUACUUCGAAUU
949
AAUUCGAAGUAUACCUCCA
950
4174
GGAGGUAUACUUCGAAUUU
951
AAAUUCGAAGUAUACCUCC
952
4175
GAGGUAUACUUCGAAUUUU
953
AAAAUUCGAAGUAUACCUC
954
4252
UUCUGGUCUGACCGUCGCA
955
UGCGACGGUCAGACCAGAA
956
4253
UCUGGUCUGACCGUCGCAA
957
UUGCGACGGUCAGACCAGA
958
4257
GUCUGACCGUCGCAACCCU
959
AGGGUUGCGACGGUCAGAC
960
4269
CAACCCUCAUGAAGUACAA
961
UUGUACUUCAUGAGGGUUG
962
4294
UAUGCUACAAGGUACGCAA
963
UUGCGUACCUUGUAGCAUA
964
4295
AUGCUACAAGGUACGCAAU
965
AUUGCGUACCUUGUAGCAU
966
4296
UGCUACAAGGUACGCAAUA
967
UAUUGCGUACCUUGUAGCA
968
4297
GCUACAAGGUACGCAAUAA
969
UUAUUGCGUACCUUGUAGC
970
4299
UACAAGGUACGCAAUAACU
971
AGUUAUUGCGUACCUUGUA
972
4306
UACGCAAUAACUGUUUGGU
973
ACCAAACAGUUAUUGCGUA
974
4307
ACGCAAUAACUGUUUGGUA
975
UACCAAACAGUUAUUGCGU
976
4335
AGAUGAGAGAGCACGAGCU
977
AGCUCGUGCUCUCUCAUCU
978
4337
AUGAGAGAGCACGAGCUAA
979
UUAGCUCGUGCUCUCUCAU
980
4340
AGAGAGCACGAGCUAAAGU
981
ACUUUAGCUCGUGCUCUCU
982
4341
GAGAGCACGAGCUAAAGUA
983
UACUUUAGCUCGUGCUCUC
984
4342
AGAGCACGAGCUAAAGUAA
985
UUACUUUAGCUCGUGCUCU
986
4356
AGUAAAAUAUCUAACAGGU
987
ACCUGUUAGAUAUUUUACU
988
4358
UAAAAUAUCUAACAGGUGA
989
UCACCUGUUAGAUAUUUUA
990
4359
AAAAUAUCUAACAGGUGAA
991
UUCACCUGUUAGAUAUUUU
992
4360
AAAUAUCUAACAGGUGAAA
993
UUUCACCUGUUAGAUAUUU
994
4379
AAGGUGUGAGGGUUGAACU
995
AGUUCAACCCUCACACCUU
996
4381
GGUGUGAGGGUUGAACUCA
997
UGAGUUCAACCCUCACACC
998
4384
GUGAGGGUUGAACUCAAUA
999
UAUUGAGUUCAACCCUCAC
1000
4386
GAGGGUUGAACUCAAUAAA
1001
UUUAUUGAGUUCAACCCUC
1002
4389
GGUUGAACUCAAUAAACCU
1003
AGGUUUAUUGAGUUCAACC
1004
4404
ACCUUCAGAUUCGGUCGGU
1005
ACCGACCGAAUCUGAAGGU
1006
4405
CCUUCAGAUUCGGUCGGUA
1007
UACCGACCGAAUCUGAAGG
1008
4406
CUUCAGAUUCGGUCGGUAA
1009
UUACCGACCGAAUCUGAAG
1010
4407
UUCAGAUUCGGUCGGUAAA
1011
UUUACCGACCGAAUCUGAA
1012
4409
CAGAUUCGGUCGGUAAAGA
1013
UCUUUACCGACCGAAUCUG
1014
4412
AUUCGGUCGGUAAAGACGU
1015
ACGUCUUUACCGACCGAAU
1016
4424
AAGACGUCUUCUAGAGCCU
1017
AGGCUCUAGAAGACGUCUU
1018
4425
AGACGUCUUCUAGAGCCUU
1019
AAGGCUCUAGAAGACGUCU
1020
4435
UAGAGCCUUUGAUCCAGCA
1021
UGCUGGAUCAAAGGCUCUA
1022
4443
UUGAUCCAGCAAUACCCCA
1023
UGGGGUAUUGCUGGAUCAA
1024
4451
GCAAUACCCCACUUCACCU
1025
AGGUGAAGUGGGGUAUUGC
1026
4461
ACUUCACCUACAAUAUUGU
1027
ACAAUAUUGUAGGUGAAGU
1028
4488
UGUUAACUUGUGAAUACGA
1029
UCGUAUUCACAAGUUAACA
1030
4489
GUUAACUUGUGAAUACGAA
1031
UUCGUAUUCACAAGUUAAC
1032
4494
CUUGUGAAUACGAAUAAAU
1033
AUUUAUUCGUAUUCACAAG
1034
4502
UACGAAUAAAUGGGAUAAA
1035
UUUAUCCCAUUUAUUCGUA
1036
4525
AAUAGACAACCAGUUCGCA
1037
UGCGAACUGGUUGUCUAUU
1038
4526
AUAGACAACCAGUUCGCAU
1039
AUGCGAACUGGUUGUCUAU
1040
4527
UAGACAACCAGUUCGCAUU
1041
AAUGCGAACUGGUUGUCUA
1042
4528
AGACAACCAGUUCGCAUUU
1043
AAAUGCGAACUGGUUGUCU
1044
4608
CUUUGUACUGCAUGAUCAA
1045
UUGAUCAUGCAGUACAAAG
1046
4634
UCUGUGAUUGCUUACAGGA
1047
UCCUGUAAGCAAUCACAGA
1048
4651
GAGGAAGAUAAGCUACUAA
1049
UUAGUAGCUUAUCUUCCUC
1050
4687
AUCUGGAUAUGAAAUAAGU
1051
ACUUAUUUCAUAUCCAGAU
1052
4699
AAUAAGUGCCCUGUGUAGA
1053
UCUACACAGGGCACUUAUU
1054
4700
AUAAGUGCCCUGUGUAGAA
1055
UUCUACACAGGGCACUUAU
1056
4703
AGUGCCCUGUGUAGAAUUU
1057
AAAUUCUACACAGGGCACU
1058
4732
UAUAUUUUGCCAGAUCUGU
1059
ACAGAUCUGGCAAAAUAUA
1060
4738
UUGCCAGAUCUGUUAUCUA
1061
UAGAUAACAGAUCUGGCAA
1062
4741
CCAGAUCUGUUAUCUAGCU
1063
AGCUAGAUAACAGAUCUGG
1064
4748
UGUUAUCUAGCUGAGUUCA
1065
UGAACUCAGCUAGAUAACA
1066
4749
GUUAUCUAGCUGAGUUCAU
1067
AUGAACUCAGCUAGAUAAC
1068
4756
AGCUGAGUUCAUUUCAUCU
1069
AGAUGAAAUGAACUCAGCU
1070
4791
AAGUUUGAAUUUGGGAUAA
1071
UUAUCCCAAAUUCAAACUU
1072
4812
UUUCUAUAUUAGGUACAAU
1073
AUUGUACCUAAUAUAGAAA
1074
4814
UCUAUAUUAGGUACAAUUU
1075
AAAUUGUACCUAAUAUAGA
1076
4819
AUUAGGUACAAUUUAUCUA
1077
UAGAUAAAUUGUACCUAAU
1078
4820
UUAGGUACAAUUUAUCUAA
1079
UUAGAUAAAUUGUACCUAA
1080
4821
UAGGUACAAUUUAUCUAAA
1081
UUUAGAUAAAUUGUACCUA
1082
4823
GGUACAAUUUAUCUAAACU
1083
AGUUUAGAUAAAUUGUACC
1084
4870
CUCAAAAUAACAUCAAUCU
1085
AGAUUGAUGUUAUUUUGAG
1086
4893
UUGUAAACCUGUUCAUACU
1087
AGUAUGAACAGGUUUACAA
1088
4894
UGUAAACCUGUUCAUACUA
1089
UAGUAUGAACAGGUUUACA
1090
4897
AAACCUGUUCAUACUAUUA
1091
UAAUAGUAUGAACAGGUUU
1092
4909
ACUAUUAAAUUUUGCCCUA
1093
UAGGGCAAAAUUUAAUAGU
1094
4919
UUUGCCCUAAAAGACCUCU
1095
AGAGGUCUUUUAGGGCAAA
1096
4920
UUGCCCUAAAAGACCUCUU
1097
AAGAGGUCUUUUAGGGCAA
1098
4929
AAGACCUCUUAAUAAUGAU
1099
AUCAUUAUUAAGAGGUCUU
1100
4930
AGACCUCUUAAUAAUGAUU
1101
AAUCAUUAUUAAGAGGUCU
1102
4933
CCUCUUAAUAAUGAUUGUU
1103
AACAAUCAUUAUUAAGAGG
1104
4952
GCCAGUGACUGAUGAUUAA
1105
UUAAUCAUCAGUCACUGGC
1106
4953
CCAGUGACUGAUGAUUAAU
1107
AUUAAUCAUCAGUCACUGG
1108
4954
CAGUGACUGAUGAUUAAUU
1109
AAUUAAUCAUCAGUCACUG
1110
4997
GAGCACUUUAAUUACAACU
1111
AGUUGUAAUUAAAGUGCUC
1112
5031
UUUGUAGUCCUUCCUUACA
1113
UGUAAGGAAGGACUACAAA
1114
5035
UAGUCCUUCCUUACACUAA
1115
UUAGUGUAAGGAAGGACUA
1116
5048
CACUAAUUUGAACUGUUAA
1117
UUAACAGUUCAAAUUAGUG
1118
5084
UUGACAUUGUCAAUAACGA
1119
UCGUUAUUGACAAUGUCAA
1120
5085
UGACAUUGUCAAUAACGAA
1121
UUCGUUAUUGACAAUGUCA
1122
5086
GACAUUGUCAAUAACGAAA
1123
UUUCGUUAUUGACAAUGUC
1124
5089
AUUGUCAAUAACGAAACCU
1125
AGGUUUCGUUAUUGACAAU
1126
5090
UUGUCAAUAACGAAACCUA
1127
UAGGUUUCGUUAUUGACAA
1128
5091
UGUCAAUAACGAAACCUAA
1129
UUAGGUUUCGUUAUUGACA
1130
5095
AAUAACGAAACCUAAUUGU
1131
ACAAUUAGGUUUCGUUAUU
1132
5096
AUAACGAAACCUAAUUGUA
1133
UACAAUUAGGUUUCGUUAU
1134
5105
CCUAAUUGUAAAACAGUCA
1135
UGACUGUUUUACAAUUAGG
1136
5111
UGUAAAACAGUCACCAUUU
1137
AAAUGGUGACUGUUUUACA
1138
5120
GUCACCAUUUACUACCAAU
1139
AUUGGUAGUAAAUGGUGAC
1140
5121
UCACCAUUUACUACCAAUA
1141
UAUUGGUAGUAAAUGGUGA
1142
5122
CACCAUUUACUACCAAUAA
1143
UUAUUGGUAGUAAAUGGUG
1144
5124
CCAUUUACUACCAAUAACU
1145
AGUUAUUGGUAGUAAAUGG
1146
5399
CCUAGGCUGGGGUUUAAGU
1147
ACUUAAACCCCAGCCUAGG
1148
5404
GCUGGGGUUUAAGUUAAAU
1149
AUUUAACUUAAACCCCAGC
1150
5405
CUGGGGUUUAAGUUAAAUU
1151
AAUUUAACUUAAACCCCAG
1152
5432
AACUAAAGUGACUGGCACU
1153
AGUGCCAGUCACUUUAGUU
1154
5474
GCUUCAAGUUCCUAAGAUA
1155
UAUCUUAGGAACUUGAAGC
1156
5481
GUUCCUAAGAUAAGGGCUU
1157
AAGCCCUUAUCUUAGGAAC
1158
5484
CCUAAGAUAAGGGCUUUCU
1159
AGAAAGCCCUUAUCUUAGG
1160
5511
CAGGUGUAUGUAUCCUCUA
1161
UAGAGGAUACAUACACCUG
1162
5513
GGUGUAUGUAUCCUCUAGA
1163
UCUAGAGGAUACAUACACC
1164
5517
UAUGUAUCCUCUAGAUGUA
1165
UACAUCUAGAGGAUACAUA
1166
5523
UCCUCUAGAUGUAGACAAU
1167
AUUGUCUACAUCUAGAGGA
1168
5524
CCUCUAGAUGUAGACAAUA
1169
UAUUGUCUACAUCUAGAGG
1170
5543
AUGUCCCAUUUCUAAGUCU
1171
AGACUUAGAAAUGGGACAU
1172
5544
UGUCCCAUUUCUAAGUCUU
1173
AAGACUUAGAAAUGGGACA
1174
5574
UCUCCUUAAAUUGAUUGUA
1175
UACAAUCAAUUUAAGGAGA
1176
5580
UAAAUUGAUUGUACUUCCA
1177
UGGAAGUACAAUCAAUUUA
1178
5581
AAAUUGAUUGUACUUCCAA
1179
UUGGAAGUACAAUCAAUUU
1180
5624
AUACUGUGAUCUAUCUGAU
1181
AUCAGAUAGAUCACAGUAU
1182
5659
UGUCUCUGUUGAAGAGCAU
1183
AUGCUCUUCAACAGAGACA
1184
5662
CUCUGUUGAAGAGCAUCAA
1185
UUGAUGCUCUUCAACAGAG
1186
5673
AGCAUCAAGGGGAGAUUAU
1187
AUAAUCUCCCCUUGAUGCU
1188
5676
AUCAAGGGGAGAUUAUGUA
1189
UACAUAAUCUCCCCUUGAU
1190
5678
CAAGGGGAGAUUAUGUACA
1191
UGUACAUAAUCUCCCCUUG
1192
5711
UGUGGUGUUACUGACGGAA
1193
UUCCGUCAGUAACACCACA
1194
5714
GGUGUUACUGACGGAAUGU
1195
ACAUUCCGUCAGUAACACC
1196
5717
GUUACUGACGGAAUGUGCA
1197
UGCACAUUCCGUCAGUAAC
1198
5723
GACGGAAUGUGCAGUAACU
1199
AGUUACUGCACAUUCCGUC
1200
5738
AACUCCUCAGAUAUCUGUU
1201
AACAGAUAUCUGAGGAGUU
1202
5740
CUCCUCAGAUAUCUGUUAA
1203
UUAACAGAUAUCUGAGGAG
1204
5782
GCCUUCUUACCUGUACUGA
1205
UCAGUACAGGUAAGAAGGC
1206
5792
CUGUACUGAAAGAUGCUUA
1207
UAAGCAUCUUUCAGUACAG
1208
5795
UACUGAAAGAUGCUUAGCU
1209
AGCUAAGCAUCUUUCAGUA
1210
5799
GAAAGAUGCUUAGCUUAGA
1211
UCUAAGCUAAGCAUCUUUC
1212
5801
AAGAUGCUUAGCUUAGAAA
1213
UUUCUAAGCUAAGCAUCUU
1214
5860
UCAUGGGUUUUCUUAUUUA
1215
UAAAUAAGAAAACCCAUGA
1216
5915
AAGGCCUCACAUACAUGUU
1217
AACAUGUAUGUGAGGCCUU
1218
5917
GGCCUCACAUACAUGUUAU
1219
AUAACAUGUAUGUGAGGCC
1220
5918
GCCUCACAUACAUGUUAUU
1221
AAUAACAUGUAUGUGAGGC
1222
5944
UGAAUUGGGACGGAUGUCU
1223
AGACAUCCGUCCCAAUUCA
1224
5945
GAAUUGGGACGGAUGUCUU
1225
AAGACAUCCGUCCCAAUUC
1226
5948
UUGGGACGGAUGUCUUAGA
1227
UCUAAGACAUCCGUCCCAA
1228
5950
GGGACGGAUGUCUUAGACU
1229
AGUCUAAGACAUCCGUCCC
1230
5961
CUUAGACUUCACUUUCCUA
1231
UAGGAAAGUGAAGUCUAAG
1232
5965
GACUUCACUUUCCUAGGCU
1233
AGCCUAGGAAAGUGAAGUC
1234
5966
ACUUCACUUUCCUAGGCUU
1235
AAGCCUAGGAAAGUGAAGU
1236
5967
CUUCACUUUCCUAGGCUUU
1237
AAAGCCUAGGAAAGUGAAG
1238
5968
UUCACUUUCCUAGGCUUUU
1239
AAAAGCCUAGGAAAGUGAA
1240
5994
ACCUAAAGGGUGGUAUCCA
1241
UGGAUACCACCCUUUAGGU
1242
5997
UAAAGGGUGGUAUCCAUAU
1243
AUAUGGAUACCACCCUUUA
1244
5998
AAAGGGUGGUAUCCAUAUU
1245
AAUAUGGAUACCACCCUUU
1246
6004
UGGUAUCCAUAUUUUGCGU
1247
ACGCAAAAUAUGGAUACCA
1248
6006
GUAUCCAUAUUUUGCGUGA
1249
UCACGCAAAAUAUGGAUAC
1250
6007
UAUCCAUAUUUUGCGUGAA
1251
UUCACGCAAAAUAUGGAUA
1252
6008
AUCCAUAUUUUGCGUGAAU
1253
AUUCACGCAAAAUAUGGAU
1254
6010
CCAUAUUUUGCGUGAAUUA
1255
UAAUUCACGCAAAAUAUGG
1256
6017
UUGCGUGAAUUAUGGGUGU
1257
ACACCCAUAAUUCACGCAA
1258
6024
AAUUAUGGGUGUAAGACCU
1259
AGGUCUUACACCCAUAAUU
1260
6025
AUUAUGGGUGUAAGACCUU
1261
AAGGUCUUACACCCAUAAU
1262
6038
GACCUUGCCCACUUAGGUU
1263
AACCUAAGUGGGCAAGGUC
1264
6048
ACUUAGGUUUUCUAUCUCU
1265
AGAGAUAGAAAACCUAAGU
1266
6050
UUAGGUUUUCUAUCUCUGU
1267
ACAGAGAUAGAAAACCUAA
1268
6057
UUCUAUCUCUGUCCUUGAU
1269
AUCAAGGACAGAGAUAGAA
1270
6059
CUAUCUCUGUCCUUGAUCU
1271
AGAUCAAGGACAGAGAUAG
1272
6083
GCCAAAAUGUGAGUAUACA
1273
UGUAUACUCACAUUUUGGC
1274
6085
CAAAAUGUGAGUAUACAGA
1275
UCUGUAUACUCACAUUUUG
1276
6137
AGCAUCUGUAUAGUUUGUA
1277
UACAAACUAUACAGAUGCU
1278
6153
GUAUUCAAUUUGAGACCUU
1279
AAGGUCUCAAAUUGAAUAC
1280
6167
ACCUUUUCUAUGGGAAGCU
1281
AGCUUCCCAUAGAAAAGGU
1282
6169
CUUUUCUAUGGGAAGCUCA
1283
UGAGCUUCCCAUAGAAAAG
1284
6206
UUGCCAUUGCUAUUCAUGU
1285
ACAUGAAUAGCAAUGGCAA
1286
6273
GGGAUUGAAUGUUCAGUAU
1287
AUACUGAACAUUCAAUCCC
1288
6290
AUAGUGAUCUCACUUAGGA
1289
UCCUAAGUGAGAUCACUAU
1290
6318
GGAGAAAGUGAUAGUUUAU
1291
AUAAACUAUCACUUUCUCC
1292
6341
UUUUCCUCGCCCAUAUUCA
1293
UGAAUAUGGGCGAGGAAAA
1294
6344
UCCUCGCCCAUAUUCAGUU
1295
AACUGAAUAUGGGCGAGGA
1296
6345
CCUCGCCCAUAUUCAGUUU
1297
AAACUGAAUAUGGGCGAGG
1298
6346
CUCGCCCAUAUUCAGUUUU
1299
AAAACUGAAUAUGGGCGAG
1300
6348
CGCCCAUAUUCAGUUUUGU
1301
ACAAAACUGAAUAUGGGCG
1302
6389
AGAUGAUAACAUCACAUCU
1303
AGAUGUGAUGUUAUCAUCU
1304
6400
UCACAUCUCUACAGUAAGU
1305
ACUUACUGUAGAGAUGUGA
1306
6431
CCAACCCAGGAGCGCAAGU
1307
ACUUGCGCUCCUGGGUUGG
1308
6432
CAACCCAGGAGCGCAAGUU
1309
AACUUGCGCUCCUGGGUUG
1310
6458
CCAUCUGGUCUAUAGUACA
1311
UGUACUAUAGACCAGAUGG
1312
6469
AUAGUACAGUGCGCGGCGU
1313
ACGCCGCGCACUGUACUAU
1314
6470
UAGUACAGUGCGCGGCGUU
1315
AACGCCGCGCACUGUACUA
1316
6471
AGUACAGUGCGCGGCGUUA
1317
UAACGCCGCGCACUGUACU
1318
6476
AGUGCGCGGCGUUAGGCCA
1319
UGGCCUAACGCCGCGCACU
1320
6478
UGCGCGGCGUUAGGCCACA
1321
UGUGGCCUAACGCCGCGCA
1322
6479
GCGCGGCGUUAGGCCACAA
1323
UUGUGGCCUAACGCCGCGC
1324
6484
GCGUUAGGCCACAACUCAA
1325
UUGAGUUGUGGCCUAACGC
1326
6485
CGUUAGGCCACAACUCAAA
1327
UUUGAGUUGUGGCCUAACG
1328
6516
UUUAGGGUUAGUAGAAAUU
1329
AAUUUCUACUAACCCUAAA
1330
6537
UUUAUGUUGAUGGGAGGUU
1331
AACCUCCCAUCAACAUAAA
1332
6548
GGGAGGUUUGUUUGAUUGU
1333
ACAAUCAAACAAACCUCCC
1334
6581
ACAGCCUUUUAAUUUGGGA
1335
UCCCAAAUUAAAAGGCUGU
1336
6599
AGCCCCUGUUGUCAUUCAA
1337
UUGAAUGACAACAGGGGCU
1338
6609
GUCAUUCAAAUGUGUACCU
1339
AGGUACACAUUUGAAUGAC
1340
6612
AUUCAAAUGUGUACCUCUA
1341
UAGAGGUACACAUUUGAAU
1342
6656
CUAUCUGUGGGUUGUGCUU
1343
AAGCACAACCCACAGAUAG
1344
6669
GUGCUUGCCAGACAGGUCU
1345
AGACCUGUCUGGCAAGCAC
1346
6716
UAUACUCUCUUAGGAAUCA
1347
UGAUUCCUAAGAGAGUAUA
1348
6747
CAAGAAAUCAGGAUGGCCA
1349
UGGCCAUCCUGAUUUCUUG
1350
6788
CAUGUUAGUGGGACUAUUA
1351
UAAUAGUCCCACUAACAUG
1352
6800
ACUAUUAACUUGUCACCAA
1353
UUGGUGACAAGUUAAUAGU
1354
6862
AUAUGUGUUUAAUCCUGGU
1355
ACCAGGAUUAAACACAUAU
1356
6868
GUUUAAUCCUGGUUAAAGA
1357
UCUUUAACCAGGAUUAAAC
1358
6869
UUUAAUCCUGGUUAAAGAU
1359
AUCUUUAACCAGGAUUAAA
1360
6911
UUCAACACAUUAACCAGCU
1361
AGCUGGUUAAUGUGUUGAA
1362
6942
CCUUUAUCAAGAGUAGGCA
1363
UGCCUACUCUUGAUAAAGG
1364
6943
CUUUAUCAAGAGUAGGCAA
1365
UUGCCUACUCUUGAUAAAG
1366
6974
UUCAUAUACAGAUAGACUA
1367
UAGUCUAUCUGUAUAUGAA
1368
6985
AUAGACUAUAAAGUCAUGU
1369
ACAUGACUUUAUAGUCUAU
1370
6986
UAGACUAUAAAGUCAUGUA
1371
UACAUGACUUUAUAGUCUA
1372
7040
CAAGUUGCUUGUAAAGCUA
1373
UAGCUUUACAAGCAACUUG
1374
7041
AAGUUGCUUGUAAAGCUAA
1375
UUAGCUUUACAAGCAACUU
1376
7045
UGCUUGUAAAGCUAAUCUA
1377
UAGAUUAGCUUUACAAGCA
1378
7046
GCUUGUAAAGCUAAUCUAA
1379
UUAGAUUAGCUUUACAAGC
1380
TABLE 6B
Human EGNL2 Single Strands and Duplex Sequences
SEQ
SEQ
ID
ID
Start
Sense Sequence (5′ to 3′)
NO.
Antisense Sequence (5′ to 3′)
NO.
64
CCACCCUGAAGGGUCCCUU
1381
AAGGGACCCUUCAGGGUGG
1382
76
GUCCCUUCCCAAGCCCUUA
1383
UAAGGGCUUGGGAAGGGAC
1384
80
CUUCCCAAGCCCUUAGGGA
1385
UCCCUAAGGGCUUGGGAAG
1386
85
CAAGCCCUUAGGGACCGCA
1387
UGCGGUCCCUAAGGGCUUG
1388
93
UAGGGACCGCAGAGGACUU
1389
AAGUCCUCUGCGGUCCCUA
1390
98
ACCGCAGAGGACUUGGGGA
1391
UCCCCAAGUCCUCUGCGGU
1392
108
ACUUGGGGACCAGCAAGCA
1393
UGCUUGCUGGUCCCCAAGU
1394
109
CUUGGGGACCAGCAAGCAA
1395
UUGCUUGCUGGUCCCCAAG
1396
115
GACCAGCAAGCAACCCCCA
1397
UGGGGGUUGCUUGCUGGUC
1398
125
CAACCCCCAGGGCACGAGA
1399
UCUCGUGCCCUGGGGGUUG
1400
126
AACCCCCAGGGCACGAGAA
1401
UUCUCGUGCCCUGGGGGUU
1402
128
CCCCCAGGGCACGAGAAGA
1403
UCUUCUCGUGCCCUGGGGG
1404
137
CACGAGAAGAGCUCUUGCU
1405
AGCAAGAGCUCUUCUCGUG
1406
139
CGAGAAGAGCUCUUGCUGU
1407
ACAGCAAGAGCUCUUCUCG
1408
141
AGAAGAGCUCUUGCUGUCU
1409
AGACAGCAAGAGCUCUUCU
1410
195
GCCCCCAGCUGCAUCAAGU
1411
ACUUGAUGCAGCUGGGGGC
1412
244
CACCAUGGGCCCGGGCGGU
1413
ACCGCCCGGGCCCAUGGUG
1414
253
CCCGGGCGGUGCCCUCCAU
1415
AUGGAGGGCACCGCCCGGG
1416
266
CUCCAUGCCCGGGGGAUGA
1417
UCAUCCCCCGGGCAUGGAG
1418
269
CAUGCCCGGGGGAUGAAGA
1419
UCUUCAUCCCCCGGGCAUG
1420
271
UGCCCGGGGGAUGAAGACA
1421
UGUCUUCAUCCCCCGGGCA
1422
273
CCCGGGGGAUGAAGACACU
1423
AGUGUCUUCAUCCCCCGGG
1424
276
GGGGGAUGAAGACACUGCU
1425
AGCAGUGUCUUCAUCCCCC
1426
310
UGCCAGCCGCAGCCCCUAA
1427
UUAGGGGCUGCGGCUGGCA
1428
314
AGCCGCAGCCCCUAAGUCA
1429
UGACUUAGGGGCUGCGGCU
1430
318
GCAGCCCCUAAGUCAGGCU
1431
AGCCUGACUUAGGGGCUGC
1432
320
AGCCCCUAAGUCAGGCUCU
1433
AGAGCCUGACUUAGGGGCU
1434
324
CCUAAGUCAGGCUCUCCCU
1435
AGGGAGAGCCUGACUUAGG
1436
328
AGUCAGGCUCUCCCUCAGU
1437
ACUGAGGGAGAGCCUGACU
1438
329
GUCAGGCUCUCCCUCAGUU
1439
AACUGAGGGAGAGCCUGAC
1440
340
CCUCAGUUACCAGGGUCUU
1441
AAGACCCUGGUAACUGAGG
1442
343
CAGUUACCAGGGUCUUCGU
1443
ACGAAGACCCUGGUAACUG
1444
345
GUUACCAGGGUCUUCGUCA
1445
UGACGAAGACCCUGGUAAC
1446
347
UACCAGGGUCUUCGUCAGA
1447
UCUGACGAAGACCCUGGUA
1448
398
UGGGAGUGGAGAGUUACCU
1449
AGGUAACUCUCCACUCCCA
1450
441
CCACUGUCCAGGAGUGCCU
1451
AGGCACUCCUGGACAGUGG
1452
456
GCCUAGUGAGGCCUCGGCA
1453
UGCCGAGGCCUCACUAGGC
1454
516
CAGCCCUCUUCGGGACGGU
1455
ACCGUCCCGAAGAGGGCUG
1456
518
GCCCUCUUCGGGACGGUUU
1457
AAACCGUCCCGAAGAGGGC
1458
519
CCCUCUUCGGGACGGUUUU
1459
AAAACCGUCCCGAAGAGGG
1460
527
GGGACGGUUUUGGCGGGCA
1461
UGCCCGCCAAAACCGUCCC
1462
531
CGGUUUUGGCGGGCAGGAU
1463
AUCCUGCCCGCCAAAACCG
1464
534
UUUUGGCGGGCAGGAUGGU
1465
ACCAUCCUGCCCGCCAAAA
1466
561
GCGGCCGCUGCAGAGUGAA
1467
UUCACUCUGCAGCGGCCGC
1468
567
GCUGCAGAGUGAAGGCGCU
1469
AGCGCCUUCACUCUGCAGC
1470
583
GCUGCAGCGCUGGUCACCA
1471
UGGUGACCAGCGCUGCAGC
1472
593
UGGUCACCAAGGGGUGCCA
1473
UGGCACCCCUUGGUGACCA
1474
598
ACCAAGGGGUGCCAGCGAU
1475
AUCGCUGGCACCCCUUGGU
1476
599
CCAAGGGGUGCCAGCGAUU
1477
AAUCGCUGGCACCCCUUGG
1478
603
GGGGUGCCAGCGAUUGGCA
1479
UGCCAAUCGCUGGCACCCC
1480
615
AUUGGCAGCCCAGGGCGCA
1481
UGCGCCCUGGGCUGCCAAU
1482
637
CCUGAGGCCCCCAAACGGA
1483
UCCGUUUGGGGGCCUCAGG
1484
638
CUGAGGCCCCCAAACGGAA
1485
UUCCGUUUGGGGGCCUCAG
1486
639
UGAGGCCCCCAAACGGAAA
1487
UUUCCGUUUGGGGGCCUCA
1488
640
GAGGCCCCCAAACGGAAAU
1489
AUUUCCGUUUGGGGGCCUC
1490
650
AACGGAAAUGGGCCGAGGA
1491
UCCUCGGCCCAUUUCCGUU
1492
651
ACGGAAAUGGGCCGAGGAU
1493
AUCCUCGGCCCAUUUCCGU
1494
654
GAAAUGGGCCGAGGAUGGU
1495
ACCAUCCUCGGCCCAUUUC
1496
685
UCACCCAGCAAACGGCCCU
1497
AGGGCCGUUUGCUGGGUGA
1498
704
GGGCCAGGCAAGAGAACCA
1499
UGGUUCUCUUGCCUGGCCC
1500
803
CGCUGCCCUCUGCGCCCGA
1501
UCGGGCGCAGAGGGCAGCG
1502
824
GCCUGGCCCUGGACUAUAU
1503
AUAUAGUCCAGGGCCAGGC
1504
827
UGGCCCUGGACUAUAUCGU
1505
ACGAUAUAGUCCAGGGCCA
1506
835
GACUAUAUCGUGCCCUGCA
1507
UGCAGGGCACGAUAUAGUC
1508
836
ACUAUAUCGUGCCCUGCAU
1509
AUGCAGGGCACGAUAUAGU
1510
842
UCGUGCCCUGCAUGCGGUA
1511
UACCGCAUGCAGGGCACGA
1512
844
GUGCCCUGCAUGCGGUACU
1513
AGUACCGCAUGCAGGGCAC
1514
845
UGCCCUGCAUGCGGUACUA
1515
UAGUACCGCAUGCAGGGCA
1516
851
GCAUGCGGUACUACGGCAU
1517
AUGCCGUAGUACCGCAUGC
1518
853
AUGCGGUACUACGGCAUCU
1519
AGAUGCCGUAGUACCGCAU
1520
857
GGUACUACGGCAUCUGCGU
1521
ACGCAGAUGCCGUAGUACC
1522
859
UACUACGGCAUCUGCGUCA
1523
UGACGCAGAUGCCGUAGUA
1524
863
ACGGCAUCUGCGUCAAGGA
1525
UCCUUGACGCAGAUGCCGU
1526
868
AUCUGCGUCAAGGACAGCU
1527
AGCUGUCCUUGACGCAGAU
1528
896
CAGCACUGGGCGGUCGCGU
1529
ACGCGACCGCCCAGUGCUG
1530
899
CACUGGGCGGUCGCGUGCU
1531
AGCACGCGACCGCCCAGUG
1532
927
GGAGGCCCUCAAACGGGGU
1533
ACCCCGUUUGAGGGCCUCC
1534
935
UCAAACGGGGUGGGCGCCU
1535
AGGCGCCCACCCCGUUUGA
1536
939
ACGGGGUGGGCGCCUGCGA
1537
UCGCAGGCGCCCACCCCGU
1538
947
GGCGCCUGCGAGACGGGCA
1539
UGCCCGUCUCGCAGGCGCC
1540
967
CUAGUGAGCCAGAGGGCGA
1541
UCGCCCUCUGGCUCACUAG
1542
968
UAGUGAGCCAGAGGGCGAU
1543
AUCGCCCUCUGGCUCACUA
1544
982
GCGAUCCCGCCGCGCAGCA
1545
UGCUGCGCGGCGGGAUCGC
1546
983
CGAUCCCGCCGCGCAGCAU
1547
AUGCUGCGCGGCGGGAUCG
1548
987
CCCGCCGCGCAGCAUCCGU
1549
ACGGAUGCUGCGCGGCGGG
1550
992
CGCGCAGCAUCCGUGGGGA
1551
UCCCCACGGAUGCUGCGCG
1552
999
CAUCCGUGGGGACCAGAUU
1553
AAUCUGGUCCCCACGGAUG
1554
1011
CCAGAUUGCCUGGGUGGAA
1555
UUCCACCCAGGCAAUCUGG
1556
1019
CCUGGGUGGAAGGCCAUGA
1557
UCAUGGCCUUCCACCCAGG
1558
1020
CUGGGUGGAAGGCCAUGAA
1559
UUCAUGGCCUUCCACCCAG
1560
1032
CCAUGAACCAGGCUGUCGA
1561
UCGACAGCCUGGUUCAUGG
1562
1033
CAUGAACCAGGCUGUCGAA
1563
UUCGACAGCCUGGUUCAUG
1564
1036
GAACCAGGCUGUCGAAGCA
1565
UGCUUCGACAGCCUGGUUC
1566
1041
AGGCUGUCGAAGCAUUGGU
1567
ACCAAUGCUUCGACAGCCU
1568
1046
GUCGAAGCAUUGGUGCCCU
1569
AGGGCACCAAUGCUUCGAC
1570
1048
CGAAGCAUUGGUGCCCUCA
1571
UGAGGGCACCAAUGCUUCG
1572
1049
GAAGCAUUGGUGCCCUCAU
1573
AUGAGGGCACCAAUGCUUC
1574
1058
GUGCCCUCAUGGCCCAUGU
1575
ACAUGGGCCAUGAGGGCAC
1576
1070
CCCAUGUGGACGCCGUCAU
1577
AUGACGGCGUCCACAUGGG
1578
1076
UGGACGCCGUCAUCCGCCA
1579
UGGCGGAUGACGGCGUCCA
1580
1078
GACGCCGUCAUCCGCCACU
1581
AGUGGCGGAUGACGGCGUC
1582
1100
CAGGGCGGCUGGGCAGCUA
1583
UAGCUGCCCAGCCGCCCUG
1584
1103
GGCGGCUGGGCAGCUAUGU
1585
ACAUAGCUGCCCAGCCGCC
1586
1106
GGCUGGGCAGCUAUGUCAU
1587
AUGACAUAGCUGCCCAGCC
1588
1117
UAUGUCAUCAACGGGCGCA
1589
UGCGCCCGUUGAUGACAUA
1590
1120
GUCAUCAACGGGCGCACCA
1591
UGGUGCGCCCGUUGAUGAC
1592
1121
UCAUCAACGGGCGCACCAA
1593
UUGGUGCGCCCGUUGAUGA
1594
1126
AACGGGCGCACCAAGGCCA
1595
UGGCCUUGGUGCGCCCGUU
1596
1137
CAAGGCCAUGGUGGCGUGU
1597
ACACGCCACCAUGGCCUUG
1598
1143
CAUGGUGGCGUGUUACCCA
1599
UGGGUAACACGCCACCAUG
1600
1148
UGGCGUGUUACCCAGGCAA
1601
UUGCCUGGGUAACACGCCA
1602
1154
GUUACCCAGGCAACGGGCU
1603
AGCCCGUUGCCUGGGUAAC
1604
1159
CCAGGCAACGGGCUCGGGU
1605
ACCCGAGCCCGUUGCCUGG
1606
1160
CAGGCAACGGGCUCGGGUA
1607
UACCCGAGCCCGUUGCCUG
1608
1163
GCAACGGGCUCGGGUACGU
1609
ACGUACCCGAGCCCGUUGC
1610
1164
CAACGGGCUCGGGUACGUA
1611
UACGUACCCGAGCCCGUUG
1612
1165
AACGGGCUCGGGUACGUAA
1613
UUACGUACCCGAGCCCGUU
1614
1169
GGCUCGGGUACGUAAGGCA
1615
UGCCUUACGUACCCGAGCC
1616
1172
UCGGGUACGUAAGGCACGU
1617
ACGUGCCUUACGUACCCGA
1618
1173
CGGGUACGUAAGGCACGUU
1619
AACGUGCCUUACGUACCCG
1620
1175
GGUACGUAAGGCACGUUGA
1621
UCAACGUGCCUUACGUACC
1622
1177
UACGUAAGGCACGUUGACA
1623
UGUCAACGUGCCUUACGUA
1624
1178
ACGUAAGGCACGUUGACAA
1625
UUGUCAACGUGCCUUACGU
1626
1179
CGUAAGGCACGUUGACAAU
1627
AUUGUCAACGUGCCUUACG
1628
1190
UUGACAAUCCCCACGGCGA
1629
UCGCCGUGGGGAUUGUCAA
1630
1191
UGACAAUCCCCACGGCGAU
1631
AUCGCCGUGGGGAUUGUCA
1632
1201
CACGGCGAUGGGCGCUGCA
1633
UGCAGCGCCCAUCGCCGUG
1634
1210
GGGCGCUGCAUCACCUGUA
1635
UACAGGUGAUGCAGCGCCC
1636
1211
GGCGCUGCAUCACCUGUAU
1637
AUACAGGUGAUGCAGCGCC
1638
1213
CGCUGCAUCACCUGUAUCU
1639
AGAUACAGGUGAUGCAGCG
1640
1216
UGCAUCACCUGUAUCUAUU
1641
AAUAGAUACAGGUGAUGCA
1642
1217
GCAUCACCUGUAUCUAUUA
1643
UAAUAGAUACAGGUGAUGC
1644
1220
UCACCUGUAUCUAUUACCU
1645
AGGUAAUAGAUACAGGUGA
1646
1222
ACCUGUAUCUAUUACCUGA
1647
UCAGGUAAUAGAUACAGGU
1648
1223
CCUGUAUCUAUUACCUGAA
1649
UUCAGGUAAUAGAUACAGG
1650
1226
GUAUCUAUUACCUGAAUCA
1651
UGAUUCAGGUAAUAGAUAC
1652
1228
AUCUAUUACCUGAAUCAGA
1653
UCUGAUUCAGGUAAUAGAU
1654
1231
UAUUACCUGAAUCAGAACU
1655
AGUUCUGAUUCAGGUAAUA
1656
1238
UGAAUCAGAACUGGGACGU
1657
ACGUCCCAGUUCUGAUUCA
1658
1239
GAAUCAGAACUGGGACGUU
1659
AACGUCCCAGUUCUGAUUC
1660
1240
AAUCAGAACUGGGACGUUA
1661
UAACGUCCCAGUUCUGAUU
1662
1241
AUCAGAACUGGGACGUUAA
1663
UUAACGUCCCAGUUCUGAU
1664
1244
AGAACUGGGACGUUAAGGU
1665
ACCUUAACGUCCCAGUUCU
1666
1247
ACUGGGACGUUAAGGUGCA
1667
UGCACCUUAACGUCCCAGU
1668
1248
CUGGGACGUUAAGGUGCAU
1669
AUGCACCUUAACGUCCCAG
1670
1256
UUAAGGUGCAUGGCGGCCU
1671
AGGCCGCCAUGCACCUUAA
1672
1259
AGGUGCAUGGCGGCCUGCU
1673
AGCAGGCCGCCAUGCACCU
1674
1294
GGCCGGCCCGUGGUAGCCA
1675
UGGCUACCACGGGCCGGCC
1676
1295
GCCGGCCCGUGGUAGCCAA
1677
UUGGCUACCACGGGCCGGC
1678
1297
CGGCCCGUGGUAGCCAACA
1679
UGUUGGCUACCACGGGCCG
1680
1298
GGCCCGUGGUAGCCAACAU
1681
AUGUUGGCUACCACGGGCC
1682
1309
GCCAACAUCGAGCCACUCU
1683
AGAGUGGCUCGAUGUUGGC
1684
1310
CCAACAUCGAGCCACUCUU
1685
AAGAGUGGCUCGAUGUUGG
1686
1311
CAACAUCGAGCCACUCUUU
1687
AAAGAGUGGCUCGAUGUUG
1688
1313
ACAUCGAGCCACUCUUUGA
1689
UCAAAGAGUGGCUCGAUGU
1690
1318
GAGCCACUCUUUGACCGGU
1691
ACCGGUCAAAGAGUGGCUC
1692
1319
AGCCACUCUUUGACCGGUU
1693
AACCGGUCAAAGAGUGGCU
1694
1322
CACUCUUUGACCGGUUGCU
1695
AGCAACCGGUCAAAGAGUG
1696
1324
CUCUUUGACCGGUUGCUCA
1697
UGAGCAACCGGUCAAAGAG
1698
1325
UCUUUGACCGGUUGCUCAU
1699
AUGAGCAACCGGUCAAAGA
1700
1326
CUUUGACCGGUUGCUCAUU
1701
AAUGAGCAACCGGUCAAAG
1702
1327
UUUGACCGGUUGCUCAUUU
1703
AAAUGAGCAACCGGUCAAA
1704
1330
GACCGGUUGCUCAUUUUCU
1705
AGAAAAUGAGCAACCGGUC
1706
1346
UCUGGUCUGACCGGCGGAA
1707
UUCCGCCGGUCAGACCAGA
1708
1352
CUGACCGGCGGAACCCCCA
1709
UGGGGGUUCCGCCGGUCAG
1710
1355
ACCGGCGGAACCCCCACGA
1711
UCGUGGGGGUUCCGCCGGU
1712
1358
GGCGGAACCCCCACGAGGU
1713
ACCUCGUGGGGGUUCCGCC
1714
1361
GGAACCCCCACGAGGUGAA
1715
UUCACCUCGUGGGGGUUCC
1716
1370
ACGAGGUGAAGCCAGCCUA
1717
UAGGCUGGCUUCACCUCGU
1718
1375
GUGAAGCCAGCCUAUGCCA
1719
UGGCAUAGGCUGGCUUCAC
1720
1381
CCAGCCUAUGCCACCAGGU
1721
ACCUGGUGGCAUAGGCUGG
1722
1387
UAUGCCACCAGGUACGCCA
1723
UGGCGUACCUGGUGGCAUA
1724
1388
AUGCCACCAGGUACGCCAU
1725
AUGGCGUACCUGGUGGCAU
1726
1394
CCAGGUACGCCAUCACUGU
1727
ACAGUGAUGGCGUACCUGG
1728
1396
AGGUACGCCAUCACUGUCU
1729
AGACAGUGAUGGCGUACCU
1730
1401
CGCCAUCACUGUCUGGUAU
1731
AUACCAGACAGUGAUGGCG
1732
1403
CCAUCACUGUCUGGUAUUU
1733
AAAUACCAGACAGUGAUGG
1734
1438
GCAGCAGCCAAAGACAAGU
1735
ACUUGUCUUUGGCUGCUGC
1736
1440
AGCAGCCAAAGACAAGUAU
1737
AUACUUGUCUUUGGCUGCU
1738
1442
CAGCCAAAGACAAGUAUCA
1739
UGAUACUUGUCUUUGGCUG
1740
1446
CAAAGACAAGUAUCAGCUA
1741
UAGCUGAUACUUGUCUUUG
1742
1449
AGACAAGUAUCAGCUAGCA
1743
UGCUAGCUGAUACUUGUCU
1744
1450
GACAAGUAUCAGCUAGCAU
1745
AUGCUAGCUGAUACUUGUC
1746
1452
CAAGUAUCAGCUAGCAUCA
1747
UGAUGCUAGCUGAUACUUG
1748
1455
GUAUCAGCUAGCAUCAGGA
1749
UCCUGAUGCUAGCUGAUAC
1750
1457
AUCAGCUAGCAUCAGGACA
1751
UGUCCUGAUGCUAGCUGAU
1752
1459
CAGCUAGCAUCAGGACAGA
1753
UCUGUCCUGAUGCUAGCUG
1754
1461
GCUAGCAUCAGGACAGAAA
1755
UUUCUGUCCUGAUGCUAGC
1756
1476
GAAAGGUGUCCAAGUACCU
1757
AGGUACUUGGACACCUUUC
1758
1482
UGUCCAAGUACCUGUAUCA
1759
UGAUACAGGUACUUGGACA
1760
1504
CCGCCUACGCCCACCUAGU
1761
ACUAGGUGGGCGUAGGCGG
1762
1509
UACGCCCACCUAGUGGCCA
1763
UGGCCACUAGGUGGGCGUA
1764
1517
CCUAGUGGCCAGUCCCAGA
1765
UCUGGGACUGGCCACUAGG
1766
1538
CGCAUGGCAGACAGCUUAA
1767
UUAAGCUGUCUGCCAUGCG
1768
1539
GCAUGGCAGACAGCUUAAA
1769
UUUAAGCUGUCUGCCAUGC
1770
1542
UGGCAGACAGCUUAAAUGA
1771
UCAUUUAAGCUGUCUGCCA
1772
1544
GCAGACAGCUUAAAUGACU
1773
AGUCAUUUAAGCUGUCUGC
1774
1674
AGGAGGAGAAGAGACCUUU
1775
AAAGGUCUCUUCUCCUCCU
1776
1684
GAGACCUUUGCUGCCCCAU
1777
AUGGGGCAGCAAAGGUCUC
1778
1702
UCAUGGGGGCUGGGGUUGU
1779
ACAACCCCAGCCCCCAUGA
1780
1741
GUGGAGGCCACCGUUACCA
1781
UGGUAACGGUGGCCUCCAC
1782
1742
UGGAGGCCACCGUUACCAA
1783
UUGGUAACGGUGGCCUCCA
1784
1744
GAGGCCACCGUUACCAACU
1785
AGUUGGUAACGGUGGCCUC
1786
1746
GGCCACCGUUACCAACUGA
1787
UCAGUUGGUAACGGUGGCC
1788
1774
CCUGGGUCCUACCCUGUCU
1789
AGACAGGGUAGGACCCAGG
1790
1779
GUCCUACCCUGUCUGGUCA
1791
UGACCAGACAGGGUAGGAC
1792
1782
CUACCCUGUCUGGUCAUGA
1793
UCAUGACCAGACAGGGUAG
1794
1787
CUGUCUGGUCAUGACCCCA
1795
UGGGGUCAUGACCAGACAG
1796
1788
UGUCUGGUCAUGACCCCAU
1797
AUGGGGUCAUGACCAGACA
1798
1789
GUCUGGUCAUGACCCCAUU
1799
AAUGGGGUCAUGACCAGAC
1800
1798
UGACCCCAUUAGGUAUGGA
1801
UCCAUACCUAAUGGGGUCA
1802
1800
ACCCCAUUAGGUAUGGAGA
1803
UCUCCAUACCUAAUGGGGU
1804
1807
UAGGUAUGGAGAGCUGGGA
1805
UCCCAGCUCUCCAUACCUA
1806
1820
CUGGGAGGAGGCAUUGUCA
1807
UGACAAUGCCUCCUCCCAG
1808
1823
GGAGGAGGCAUUGUCACUU
1809
AAGUGACAAUGCCUCCUCC
1810
1827
GAGGCAUUGUCACUUCCCA
1811
UGGGAAGUGACAAUGCCUC
1812
1830
GCAUUGUCACUUCCCACCA
1813
UGGUGGGAAGUGACAAUGC
1814
1856
GGACUUGGGGUUGAGGUGA
1815
UCACCUCAACCCCAAGUCC
1816
1858
ACUUGGGGUUGAGGUGAGU
1817
ACUCACCUCAACCCCAAGU
1818
1861
UGGGGUUGAGGUGAGUCAU
1819
AUGACUCACCUCAACCCCA
1820
1866
UUGAGGUGAGUCAUGGCCU
1821
AGGCCAUGACUCACCUCAA
1822
1868
GAGGUGAGUCAUGGCCUCU
1823
AGAGGCCAUGACUCACCUC
1824
1872
UGAGUCAUGGCCUCUUGCU
1825
AGCAAGAGGCCAUGACUCA
1826
1876
UCAUGGCCUCUUGCUGGCA
1827
UGCCAGCAAGAGGCCAUGA
1828
1878
AUGGCCUCUUGCUGGCAAU
1829
AUUGCCAGCAAGAGGCCAU
1830
1883
CUCUUGCUGGCAAUGGGGU
1831
ACCCCAUUGCCAGCAAGAG
1832
1893
CAAUGGGGUGGGAGGAGUA
1833
UACUCCUCCCACCCCAUUG
1834
1902
GGGAGGAGUACCCCCAAGU
1835
ACUUGGGGGUACUCCUCCC
1836
1905
AGGAGUACCCCCAAGUCCU
1837
AGGACUUGGGGGUACUCCU
1838
1931
CUCCAGCCUGGAAUGUGAA
1839
UUCACAUUCCAGGCUGGAG
1840
1933
CCAGCCUGGAAUGUGAAGU
1841
ACUUCACAUUCCAGGCUGG
1842
1942
AAUGUGAAGUGACUCCCCA
1843
UGGGGAGUCACUUCACAUU
1844
1964
CCUUUGGCCAUGGCAGGCA
1845
UGCCUGCCAUGGCCAAAGG
1846
1973
AUGGCAGGCACCUUUUGGA
1847
UCCAAAAGGUGCCUGCCAU
1848
1980
GCACCUUUUGGACUGGGCU
1849
AGCCCAGUCCAAAAGGUGC
1850
2001
CACUGCUUGGGCAGAGUAA
1851
UUACUCUGCCCAAGCAGUG
1852
2002
ACUGCUUGGGCAGAGUAAA
1853
UUUACUCUGCCCAAGCAGU
1854
2003
CUGCUUGGGCAGAGUAAAA
1855
UUUUACUCUGCCCAAGCAG
1856
2006
CUUGGGCAGAGUAAAAGGU
1857
ACCUUUUACUCUGCCCAAG
1858
2010
GGCAGAGUAAAAGGUGCCA
1859
UGGCACCUUUUACUCUGCC
1860
2077
CCUCAGAGCUGCAAAAAAA
1861
UUUUUUUGCAGCUCUGAGG
1862
TABLE 6C
Human EGNL3 Single Strands and Duplex Sequences
SEQ
SEQ
Sense
ID
Antisense
ID
Start
Sequence (5′ to 3′)
NO.
Sequence (5′ to 3′)
NO.
6
UGGCCGCAGUCGCGGCAGU
1863
ACUGCCGCGACUGCGGCCA
1864
35
CAUCCCCAAAAGGCGCCCU
1865
AGGGCGCCUUUUGGGGAUG
1866
41
CAAAAGGCGCCCUCCGACU
1867
AGUCGGAGGGCGCCUUUUG
1868
53
UCCGACUCCUUGCGCCGCA
1869
UGCGGCGCAAGGAGUCGGA
1870
58
CUCCUUGCGCCGCACUGCU
1871
AGCAGUGCGGCGCAAGGAG
1872
75
CUCGCCGGGCCAGUCCGGA
1873
UCCGGACUGGCCCGGCGAG
1874
76
UCGCCGGGCCAGUCCGGAA
1875
UUCCGGACUGGCCCGGCGA
1876
77
CGCCGGGCCAGUCCGGAAA
1877
UUUCCGGACUGGCCCGGCG
1878
85
CAGUCCGGAAACGGGUCGU
1879
ACGACCCGUUUCCGGACUG
1880
88
UCCGGAAACGGGUCGUGGA
1881
UCCACGACCCGUUUCCGGA
1882
99
GUCGUGGAGCUCCGCACCA
1883
UGGUGCGGAGCUCCACGAC
1884
101
CGUGGAGCUCCGCACCACU
1885
AGUGGUGCGGAGCUCCACG
1886
107
GCUCCGCACCACUCCCGCU
1887
AGCGGGAGUGGUGCGGAGC
1888
111
CGCACCACUCCCGCUGGUU
1889
AACCAGCGGGAGUGGUGCG
1890
123
GCUGGUUCCCGAAGGCAGA
1891
UCUGCCUUCGGGAACCAGC
1892
129
UCCCGAAGGCAGAUCCCUU
1893
AAGGGAUCUGCCUUCGGGA
1894
138
CAGAUCCCUUCUCCCGAGA
1895
UCUCGGGAGAAGGGAUCUG
1896
140
GAUCCCUUCUCCCGAGAGU
1897
ACUCUCGGGAGAAGGGAUC
1898
141
AUCCCUUCUCCCGAGAGUU
1899
AACUCUCGGGAGAAGGGAU
1900
145
CUUCUCCCGAGAGUUGCGA
1901
UCGCAACUCUCGGGAGAAG
1902
147
UCUCCCGAGAGUUGCGAGA
1903
UCUCGCAACUCUCGGGAGA
1904
148
CUCCCGAGAGUUGCGAGAA
1905
UUCUCGCAACUCUCGGGAG
1906
149
UCCCGAGAGUUGCGAGAAA
1907
UUUCUCGCAACUCUCGGGA
1908
151
CCGAGAGUUGCGAGAAACU
1909
AGUUUCUCGCAACUCUCGG
1910
152
CGAGAGUUGCGAGAAACUU
1911
AAGUUUCUCGCAACUCUCG
1912
153
GAGAGUUGCGAGAAACUUU
1913
AAAGUUUCUCGCAACUCUC
1914
158
UUGCGAGAAACUUUCCCUU
1915
AAGGGAAAGUUUCUCGCAA
1916
160
GCGAGAAACUUUCCCUUGU
1917
ACAAGGGAAAGUUUCUCGC
1918
189
GCAGCGGCUCGGGUACCGU
1919
ACGGUACCCGAGCCGCUGC
1920
206
GUGGCAGCCGCAGGUUUCU
1921
AGAAACCUGCGGCUGCCAC
1922
208
GGCAGCCGCAGGUUUCUGA
1923
UCAGAAACCUGCGGCUGCC
1924
209
GCAGCCGCAGGUUUCUGAA
1925
UUCAGAAACCUGCGGCUGC
1926
245
CGCGCCUCGGCUUCGCGCU
1927
AGCGCGAAGCCGAGGCGCG
1928
250
CUCGGCUUCGCGCUCGUGU
1929
ACACGAGCGCGAAGCCGAG
1930
251
UCGGCUUCGCGCUCGUGUA
1931
UACACGAGCGCGAAGCCGA
1932
253
GGCUUCGCGCUCGUGUAGA
1933
UCUACACGAGCGCGAAGCC
1934
254
GCUUCGCGCUCGUGUAGAU
1935
AUCUACACGAGCGCGAAGC
1936
257
UCGCGCUCGUGUAGAUCGU
1937
ACGAUCUACACGAGCGCGA
1938
258
CGCGCUCGUGUAGAUCGUU
1939
AACGAUCUACACGAGCGCG
1940
262
CUCGUGUAGAUCGUUCCCU
1941
AGGGAACGAUCUACACGAG
1942
270
GAUCGUUCCCUCUCUGGUU
1943
AACCAGAGAGGGAACGAUC
1944
273
CGUUCCCUCUCUGGUUGCA
1945
UGCAACCAGAGAGGGAACG
1946
277
CCCUCUCUGGUUGCACGCU
1947
AGCGUGCAACCAGAGAGGG
1948
282
UCUGGUUGCACGCUGGGGA
1949
UCCCCAGCGUGCAACCAGA
1950
283
CUGGUUGCACGCUGGGGAU
1951
AUCCCCAGCGUGCAACCAG
1952
295
UGGGGAUCCCGGACCUCGA
1953
UCGAGGUCCGGGAUCCCCA
1954
296
GGGGAUCCCGGACCUCGAU
1955
AUCGAGGUCCGGGAUCCCC
1956
299
GAUCCCGGACCUCGAUUCU
1957
AGAAUCGAGGUCCGGGAUC
1958
307
ACCUCGAUUCUGCGGGCGA
1959
UCGCCCGCAGAAUCGAGGU
1960
309
CUCGAUUCUGCGGGCGAGA
1961
UCUCGCCCGCAGAAUCGAG
1962
355
ACCUGGAGAAAAUUGCCCU
1963
AGGGCAAUUUUCUCCAGGU
1964
367
UUGCCCUGGAGUACAUCGU
1965
ACGAUGUACUCCAGGGCAA
1966
376
AGUACAUCGUGCCCUGUCU
1967
AGACAGGGCACGAUGUACU
1968
382
UCGUGCCCUGUCUGCACGA
1969
UCGUGCAGACAGGGCACGA
1970
390
UGUCUGCACGAGGUGGGCU
1971
AGCCCACCUCGUGCAGACA
1972
451
GCGUCCUGGAGCGCGUCAA
1973
UUGACGCGCUCCAGGACGC
1974
521
CGCCGGCGUCUCCAAGCGA
1975
UCGCUUGGAGACGCCGGCG
1976
526
GCGUCUCCAAGCGACACCU
1977
AGGUGUCGCUUGGAGACGC
1978
538
GACACCUGCGGGGCGACCA
1979
UGGUCGCCCCGCAGGUGUC
1980
540
CACCUGCGGGGCGACCAGA
1981
UCUGGUCGCCCCGCAGGUG
1982
559
UCACGUGGAUCGGGGGCAA
1983
UUGCCCCCGAUCCACGUGA
1984
565
GGAUCGGGGGCAACGAGGA
1985
UCCUCGUUGCCCCCGAUCC
1986
619
UCGACAGGCUGGUCCUCUA
1987
UAGAGGACCAGCCUGUCGA
1988
621
GACAGGCUGGUCCUCUACU
1989
AGUAGAGGACCAGCCUGUC
1990
627
CUGGUCCUCUACUGCGGGA
1991
UCCCGCAGUAGAGGACCAG
1992
643
GGAGCCGGCUGGGCAAAUA
1993
UAUUUGCCCAGCCGGCUCC
1994
646
GCCGGCUGGGCAAAUACUA
1995
UAGUAUUUGCCCAGCCGGC
1996
649
GGCUGGGCAAAUACUACGU
1997
ACGUAGUAUUUGCCCAGCC
1998
651
CUGGGCAAAUACUACGUCA
1999
UGACGUAGUAUUUGCCCAG
2000
652
UGGGCAAAUACUACGUCAA
2001
UUGACGUAGUAUUUGCCCA
2002
655
GCAAAUACUACGUCAAGGA
2003
UCCUUGACGUAGUAUUUGC
2004
662
CUACGUCAAGGAGAGGUCU
2005
AGACCUCUCCUUGACGUAG
2006
663
UACGUCAAGGAGAGGUCUA
2007
UAGACCUCUCCUUGACGUA
2008
668
CAAGGAGAGGUCUAAGGCA
2009
UGCCUUAGACCUCUCCUUG
2010
673
AGAGGUCUAAGGCAAUGGU
2011
ACCAUUGCCUUAGACCUCU
2012
678
UCUAAGGCAAUGGUGGCUU
2013
AAGCCACCAUUGCCUUAGA
2014
681
AAGGCAAUGGUGGCUUGCU
2015
AGCAAGCCACCAUUGCCUU
2016
682
AGGCAAUGGUGGCUUGCUA
2017
UAGCAAGCCACCAUUGCCU
2018
683
GGCAAUGGUGGCUUGCUAU
2019
AUAGCAAGCCACCAUUGCC
2020
690
GUGGCUUGCUAUCCGGGAA
2021
UUCCCGGAUAGCAAGCCAC
2022
691
UGGCUUGCUAUCCGGGAAA
2023
UUUCCCGGAUAGCAAGCCA
2024
692
GGCUUGCUAUCCGGGAAAU
2025
AUUUCCCGGAUAGCAAGCC
2026
696
UGCUAUCCGGGAAAUGGAA
2027
UUCCAUUUCCCGGAUAGCA
2028
702
CCGGGAAAUGGAACAGGUU
2029
AACCUGUUCCAUUUCCCGG
2030
704
GGGAAAUGGAACAGGUUAU
2031
AUAACCUGUUCCAUUUCCC
2032
712
GAACAGGUUAUGUUCGCCA
2033
UGGCGAACAUAACCUGUUC
2034
715
CAGGUUAUGUUCGCCACGU
2035
ACGUGGCGAACAUAACCUG
2036
718
GUUAUGUUCGCCACGUGGA
2037
UCCACGUGGCGAACAUAAC
2038
720
UAUGUUCGCCACGUGGACA
2039
UGUCCACGUGGCGAACAUA
2040
721
AUGUUCGCCACGUGGACAA
2041
UUGUCCACGUGGCGAACAU
2042
726
CGCCACGUGGACAACCCCA
2043
UGGGGUUGUCCACGUGGCG
2044
731
CGUGGACAACCCCAACGGU
2045
ACCGUUGGGGUUGUCCACG
2046
734
GGACAACCCCAACGGUGAU
2047
AUCACCGUUGGGGUUGUCC
2048
737
CAACCCCAACGGUGAUGGU
2049
ACCAUCACCGUUGGGGUUG
2050
741
CCCAACGGUGAUGGUCGCU
2051
AGCGACCAUCACCGUUGGG
2052
744
AACGGUGAUGGUCGCUGCA
2053
UGCAGCGACCAUCACCGUU
2054
765
ACCUGCAUCUACUAUCUGA
2055
UCAGAUAGUAGAUGCAGGU
2056
766
CCUGCAUCUACUAUCUGAA
2057
UUCAGAUAGUAGAUGCAGG
2058
787
AGAAUUGGGAUGCCAAGCU
2059
AGCUUGGCAUCCCAAUUCU
2060
788
GAAUUGGGAUGCCAAGCUA
2061
UAGCUUGGCAUCCCAAUUC
2062
790
AUUGGGAUGCCAAGCUACA
2063
UGUAGCUUGGCAUCCCAAU
2064
802
AGCUACAUGGUGGGAUCCU
2065
AGGAUCCCACCAUGUAGCU
2066
808
AUGGUGGGAUCCUGCGGAU
2067
AUCCGCAGGAUCCCACCAU
2068
809
UGGUGGGAUCCUGCGGAUA
2069
UAUCCGCAGGAUCCCACCA
2070
810
GGUGGGAUCCUGCGGAUAU
2071
AUAUCCGCAGGAUCCCACC
2072
811
GUGGGAUCCUGCGGAUAUU
2073
AAUAUCCGCAGGAUCCCAC
2074
812
UGGGAUCCUGCGGAUAUUU
2075
AAAUAUCCGCAGGAUCCCA
2076
815
GAUCCUGCGGAUAUUUCCA
2077
UGGAAAUAUCCGCAGGAUC
2078
817
UCCUGCGGAUAUUUCCAGA
2079
UCUGGAAAUAUCCGCAGGA
2080
822
CGGAUAUUUCCAGAGGGGA
2081
UCCCCUCUGGAAAUAUCCG
2082
833
AGAGGGGAAAUCAUUCAUA
2083
UAUGAAUGAUUUCCCCUCU
2084
836
GGGGAAAUCAUUCAUAGCA
2085
UGCUAUGAAUGAUUUCCCC
2086
839
GAAAUCAUUCAUAGCAGAU
2087
AUCUGCUAUGAAUGAUUUC
2088
858
GUGGAGCCCAUUUUUGACA
2089
UGUCAAAAAUGGGCUCCAC
2090
860
GGAGCCCAUUUUUGACAGA
2091
UCUGUCAAAAAUGGGCUCC
2092
862
AGCCCAUUUUUGACAGACU
2093
AGUCUGUCAAAAAUGGGCU
2094
868
UUUUUGACAGACUCCUGUU
2095
AACAGGAGUCUGUCAAAAA
2096
871
UUGACAGACUCCUGUUCUU
2097
AAGAACAGGAGUCUGUCAA
2098
873
GACAGACUCCUGUUCUUCU
2099
AGAAGAACAGGAGUCUGUC
2100
881
CCUGUUCUUCUGGUCAGAU
2101
AUCUGACCAGAAGAACAGG
2102
884
GUUCUUCUGGUCAGAUCGU
2103
ACGAUCUGACCAGAAGAAC
2104
885
UUCUUCUGGUCAGAUCGUA
2105
UACGAUCUGACCAGAAGAA
2106
888
UUCUGGUCAGAUCGUAGGA
2107
UCCUACGAUCUGACCAGAA
2108
889
UCUGGUCAGAUCGUAGGAA
2109
UUCCUACGAUCUGACCAGA
2110
893
GUCAGAUCGUAGGAACCCA
2111
UGGGUUCCUACGAUCUGAC
2112
895
CAGAUCGUAGGAACCCACA
2113
UGUGGGUUCCUACGAUCUG
2114
898
AUCGUAGGAACCCACACGA
2115
UCGUGUGGGUUCCUACGAU
2116
899
UCGUAGGAACCCACACGAA
2117
UUCGUGUGGGUUCCUACGA
2118
901
GUAGGAACCCACACGAAGU
2119
ACUUCGUGUGGGUUCCUAC
2120
904
GGAACCCACACGAAGUGCA
2121
UGCACUUCGUGUGGGUUCC
2122
917
AGUGCAGCCCUCUUACGCA
2123
UGCGUAAGAGGGCUGCACU
2124
918
GUGCAGCCCUCUUACGCAA
2125
UUGCGUAAGAGGGCUGCAC
2126
921
CAGCCCUCUUACGCAACCA
2127
UGGUUGCGUAAGAGGGCUG
2128
923
GCCCUCUUACGCAACCAGA
2129
UCUGGUUGCGUAAGAGGGC
2130
926
CUCUUACGCAACCAGAUAU
2131
AUAUCUGGUUGCGUAAGAG
2132
929
UUACGCAACCAGAUAUGCU
2133
AGCAUAUCUGGUUGCGUAA
2134
933
GCAACCAGAUAUGCUAUGA
2135
UCAUAGCAUAUCUGGUUGC
2136
935
AACCAGAUAUGCUAUGACU
2137
AGUCAUAGCAUAUCUGGUU
2138
937
CCAGAUAUGCUAUGACUGU
2139
ACAGUCAUAGCAUAUCUGG
2140
942
UAUGCUAUGACUGUCUGGU
2141
ACCAGACAGUCAUAGCAUA
2142
943
AUGCUAUGACUGUCUGGUA
2143
UACCAGACAGUCAUAGCAU
2144
946
CUAUGACUGUCUGGUACUU
2145
AAGUACCAGACAGUCAUAG
2146
955
UCUGGUACUUUGAUGCUGA
2147
UCAGCAUCAAAGUACCAGA
2148
974
AGAAAGGGCAGAAGCCAAA
2149
UUUGGCUUCUGCCCUUUCU
2150
978
AGGGCAGAAGCCAAAAAGA
2151
UCUUUUUGGCUUCUGCCCU
2152
995
GAAAUUCAGGAAUUUAACU
2153
AGUUAAAUUCCUGAAUUUC
2154
996
AAAUUCAGGAAUUUAACUA
2155
UAGUUAAAUUCCUGAAUUU
2156
999
UUCAGGAAUUUAACUAGGA
2157
UCCUAGUUAAAUUCCUGAA
2158
1000
UCAGGAAUUUAACUAGGAA
2159
UUCCUAGUUAAAUUCCUGA
2160
1002
AGGAAUUUAACUAGGAAAA
2161
UUUUCCUAGUUAAAUUCCU
2162
1007
UUUAACUAGGAAAACUGAA
2163
UUCAGUUUUCCUAGUUAAA
2164
1015
GGAAAACUGAAUCUGCCCU
2165
AGGGCAGAUUCAGUUUUCC
2166
1019
AACUGAAUCUGCCCUCACU
2167
AGUGAGGGCAGAUUCAGUU
2168
1022
UGAAUCUGCCCUCACUGAA
2169
UUCAGUGAGGGCAGAUUCA
2170
1032
CUCACUGAAGACUGACCGU
2171
ACGGUCAGUCUUCAGUGAG
2172
1037
UGAAGACUGACCGUGCUCU
2173
AGAGCACGGUCAGUCUUCA
2174
1039
AAGACUGACCGUGCUCUGA
2175
UCAGAGCACGGUCAGUCUU
2176
1040
AGACUGACCGUGCUCUGAA
2177
UUCAGAGCACGGUCAGUCU
2178
1044
UGACCGUGCUCUGAAAUCU
2179
AGAUUUCAGAGCACGGUCA
2180
1052
CUCUGAAAUCUGCUGGCCU
2181
AGGCCAGCAGAUUUCAGAG
2182
1053
UCUGAAAUCUGCUGGCCUU
2183
AAGGCCAGCAGAUUUCAGA
2184
1060
UCUGCUGGCCUUGUUCAUU
2185
AAUGAACAAGGCCAGCAGA
2186
1062
UGCUGGCCUUGUUCAUUUU
2187
AAAAUGAACAAGGCCAGCA
2188
1071
UGUUCAUUUUAGUAACGGU
2189
ACCGUUACUAAAAUGAACA
2190
1072
GUUCAUUUUAGUAACGGUU
2191
AACCGUUACUAAAAUGAAC
2192
1075
CAUUUUAGUAACGGUUCCU
2193
AGGAACCGUUACUAAAAUG
2194
1078
UUUAGUAACGGUUCCUGAA
2195
UUCAGGAACCGUUACUAAA
2196
1079
UUAGUAACGGUUCCUGAAU
2197
AUUCAGGAACCGUUACUAA
2198
1080
UAGUAACGGUUCCUGAAUU
2199
AAUUCAGGAACCGUUACUA
2200
1082
GUAACGGUUCCUGAAUUCU
2201
AGAAUUCAGGAACCGUUAC
2202
1084
AACGGUUCCUGAAUUCUCU
2203
AGAGAAUUCAGGAACCGUU
2204
1088
GUUCCUGAAUUCUCUUAAA
2205
UUUAAGAGAAUUCAGGAAC
2206
1092
CUGAAUUCUCUUAAAUUCU
2207
AGAAUUUAAGAGAAUUCAG
2208
1112
UGAGAUCCAAAGAUGGCCU
2209
AGGCCAUCUUUGGAUCUCA
2210
1115
GAUCCAAAGAUGGCCUCUU
2211
AAGAGGCCAUCUUUGGAUC
2212
1119
CAAAGAUGGCCUCUUCAGU
2213
ACUGAAGAGGCCAUCUUUG
2214
1137
UGACAACAAUCUCCCUGCU
2215
AGCAGGGAGAUUGUUGUCA
2216
1141
AACAAUCUCCCUGCUACUU
2217
AAGUAGCAGGGAGAUUGUU
2218
1148
UCCCUGCUACUUCUUGCAU
2219
AUGCAAGAAGUAGCAGGGA
2220
1151
CUGCUACUUCUUGCAUCCU
2221
AGGAUGCAAGAAGUAGCAG
2222
1152
UGCUACUUCUUGCAUCCUU
2223
AAGGAUGCAAGAAGUAGCA
2224
1176
CCCUGUCUUGUGUGUGGUA
2225
UACCACACACAAGACAGGG
2226
1181
UCUUGUGUGUGGUACUUCA
2227
UGAAGUACCACACACAAGA
2228
1182
CUUGUGUGUGGUACUUCAU
2229
AUGAAGUACCACACACAAG
2230
1186
UGUGUGGUACUUCAUGUUU
2231
AAACAUGAAGUACCACACA
2232
1194
ACUUCAUGUUUUCUUGCCA
2233
UGGCAAGAAAACAUGAAGU
2234
1201
GUUUUCUUGCCAAGACUGU
2235
ACAGUCUUGGCAAGAAAAC
2236
1204
UUCUUGCCAAGACUGUGUU
2237
AACACAGUCUUGGCAAGAA
2238
1218
GUGUUGAUCUUCAGAUACU
2239
AGUAUCUGAAGAUCAACAC
2240
1222
UGAUCUUCAGAUACUCUCU
2241
AGAGAGUAUCUGAAGAUCA
2242
1228
UCAGAUACUCUCUUUGCCA
2243
UGGCAAAGAGAGUAUCUGA
2244
1230
AGAUACUCUCUUUGCCAGA
2245
UCUGGCAAAGAGAGUAUCU
2246
1233
UACUCUCUUUGCCAGAUGA
2247
UCAUCUGGCAAAGAGAGUA
2248
1234
ACUCUCUUUGCCAGAUGAA
2249
UUCAUCUGGCAAAGAGAGU
2250
1241
UUGCCAGAUGAAGUUACUU
2251
AAGUAACUUCAUCUGGCAA
2252
1245
CAGAUGAAGUUACUUGCUA
2253
UAGCAAGUAACUUCAUCUG
2254
1246
AGAUGAAGUUACUUGCUAA
2255
UUAGCAAGUAACUUCAUCU
2256
1248
AUGAAGUUACUUGCUAACU
2257
AGUUAGCAAGUAACUUCAU
2258
1251
AAGUUACUUGCUAACUCCA
2259
UGGAGUUAGCAAGUAACUU
2260
1255
UACUUGCUAACUCCAGAAA
2261
UUUCUGGAGUUAGCAAGUA
2262
1260
GCUAACUCCAGAAAUUCCU
2263
AGGAAUUUCUGGAGUUAGC
2264
1272
AAUUCCUGCAGACAUCCUA
2265
UAGGAUGUCUGCAGGAAUU
2266
1274
UUCCUGCAGACAUCCUACU
2267
AGUAGGAUGUCUGCAGGAA
2268
1287
CCUACUCGGCCAGCGGUUU
2269
AAACCGCUGGCCGAGUAGG
2270
1288
CUACUCGGCCAGCGGUUUA
2271
UAAACCGCUGGCCGAGUAG
2272
1291
CUCGGCCAGCGGUUUACCU
2273
AGGUAAACCGCUGGCCGAG
2274
1294
GGCCAGCGGUUUACCUGAU
2275
AUCAGGUAAACCGCUGGCC
2276
1295
GCCAGCGGUUUACCUGAUA
2277
UAUCAGGUAAACCGCUGGC
2278
1297
CAGCGGUUUACCUGAUAGA
2279
UCUAUCAGGUAAACCGCUG
2280
1298
AGCGGUUUACCUGAUAGAU
2281
AUCUAUCAGGUAAACCGCU
2282
1299
GCGGUUUACCUGAUAGAUU
2283
AAUCUAUCAGGUAAACCGC
2284
1303
UUUACCUGAUAGAUUCGGU
2285
ACCGAAUCUAUCAGGUAAA
2286
1304
UUACCUGAUAGAUUCGGUA
2287
UACCGAAUCUAUCAGGUAA
2288
1305
UACCUGAUAGAUUCGGUAA
2289
UUACCGAAUCUAUCAGGUA
2290
1306
ACCUGAUAGAUUCGGUAAU
2291
AUUACCGAAUCUAUCAGGU
2292
1307
CCUGAUAGAUUCGGUAAUA
2293
UAUUACCGAAUCUAUCAGG
2294
1309
UGAUAGAUUCGGUAAUACU
2295
AGUAUUACCGAAUCUAUCA
2296
1310
GAUAGAUUCGGUAAUACUA
2297
UAGUAUUACCGAAUCUAUC
2298
1313
AGAUUCGGUAAUACUAUCA
2299
UGAUAGUAUUACCGAAUCU
2300
1325
ACUAUCAAGAGAAGAGCCU
2301
AGGCUCUUCUCUUGAUAGU
2302
1329
UCAAGAGAAGAGCCUAGGA
2303
UCCUAGGCUCUUCUCUUGA
2304
1344
AGGAGCACAGCGAGGGAAU
2305
AUUCCCUCGCUGUGCUCCU
2306
1346
GAGCACAGCGAGGGAAUGA
2307
UCAUUCCCUCGCUGUGCUC
2308
1347
AGCACAGCGAGGGAAUGAA
2309
UUCAUUCCCUCGCUGUGCU
2310
1350
ACAGCGAGGGAAUGAACCU
2311
AGGUUCAUUCCCUCGCUGU
2312
1351
CAGCGAGGGAAUGAACCUU
2313
AAGGUUCAUUCCCUCGCUG
2314
1352
AGCGAGGGAAUGAACCUUA
2315
UAAGGUUCAUUCCCUCGCU
2316
1360
AAUGAACCUUACUUGCACU
2317
AGUGCAAGUAAGGUUCAUU
2318
1361
AUGAACCUUACUUGCACUU
2319
AAGUGCAAGUAAGGUUCAU
2320
1362
UGAACCUUACUUGCACUUU
2321
AAAGUGCAAGUAAGGUUCA
2322
1367
CUUACUUGCACUUUAUGUA
2323
UACAUAAAGUGCAAGUAAG
2324
1368
UUACUUGCACUUUAUGUAU
2325
AUACAUAAAGUGCAAGUAA
2326
1375
CACUUUAUGUAUACUUCCU
2327
AGGAAGUAUACAUAAAGUG
2328
1378
UUUAUGUAUACUUCCUGAU
2329
AUCAGGAAGUAUACAUAAA
2330
1379
UUAUGUAUACUUCCUGAUU
2331
AAUCAGGAAGUAUACAUAA
2332
1383
GUAUACUUCCUGAUUUGAA
2333
UUCAAAUCAGGAAGUAUAC
2334
1384
UAUACUUCCUGAUUUGAAA
2335
UUUCAAAUCAGGAAGUAUA
2336
1395
AUUUGAAAGGAGGAGGUUU
2337
AAACCUCCUCCUUUCAAAU
2338
1397
UUGAAAGGAGGAGGUUUGA
2339
UCAAACCUCCUCCUUUCAA
2340
1419
GAAAAAAAUGGAGGUGGUA
2341
UACCACCUCCAUUUUUUUC
2342
1422
AAAAAUGGAGGUGGUAGAU
2343
AUCUACCACCUCCAUUUUU
2344
1428
GGAGGUGGUAGAUGCCACA
2345
UGUGGCAUCUACCACCUCC
2346
1436
UAGAUGCCACAGAGAGGCA
2347
UGCCUCUCUGUGGCAUCUA
2348
1443
CACAGAGAGGCAUCACGGA
2349
UCCGUGAUGCCUCUCUGUG
2350
1451
GGCAUCACGGAAGCCUUAA
2351
UUAAGGCUUCCGUGAUGCC
2352
1453
CAUCACGGAAGCCUUAACA
2353
UGUUAAGGCUUCCGUGAUG
2354
1456
CACGGAAGCCUUAACAGCA
2355
UGCUGUUAAGGCUUCCGUG
2356
1476
GAAACAGAGAAAUUUGUGU
2357
ACACAAAUUUCUCUGUUUC
2358
1487
AUUUGUGUCAUCUGAACAA
2359
UUGUUCAGAUGACACAAAU
2360
1499
UGAACAAUUUCCAGAUGUU
2361
AACAUCUGGAAAUUGUUCA
2362
1501
AACAAUUUCCAGAUGUUCU
2363
AGAACAUCUGGAAAUUGUU
2364
1502
ACAAUUUCCAGAUGUUCUU
2365
AAGAACAUCUGGAAAUUGU
2366
1504
AAUUUCCAGAUGUUCUUAA
2367
UUAAGAACAUCUGGAAAUU
2368
1513
AUGUUCUUAAUCCAGGGCU
2369
AGCCCUGGAUUAAGAACAU
2370
1534
UGGGGUUUCUGGAGAAUUA
2371
UAAUUCUCCAGAAACCCCA
2372
1539
UUUCUGGAGAAUUAUCACA
2373
UGUGAUAAUUCUCCAGAAA
2374
1543
UGGAGAAUUAUCACAACCU
2375
AGGUUGUGAUAAUUCUCCA
2376
1544
GGAGAAUUAUCACAACCUA
2377
UAGGUUGUGAUAAUUCUCC
2378
1545
GAGAAUUAUCACAACCUAA
2379
UUAGGUUGUGAUAAUUCUC
2380
1546
AGAAUUAUCACAACCUAAU
2381
AUUAGGUUGUGAUAAUUCU
2382
1548
AAUUAUCACAACCUAAUGA
2383
UCAUUAGGUUGUGAUAAUU
2384
1552
AUCACAACCUAAUGACAUU
2385
AAUGUCAUUAGGUUGUGAU
2386
1553
UCACAACCUAAUGACAUUA
2387
UAAUGUCAUUAGGUUGUGA
2388
1559
CCUAAUGACAUUAAUACCU
2389
AGGUAUUAAUGUCAUUAGG
2390
1561
UAAUGACAUUAAUACCUCU
2391
AGAGGUAUUAAUGUCAUUA
2392
1565
GACAUUAAUACCUCUAGAA
2393
UUCUAGAGGUAUUAAUGUC
2394
1571
AAUACCUCUAGAAAGGGCU
2395
AGCCCUUUCUAGAGGUAUU
2396
1582
AAAGGGCUGCUGUCAUAGU
2397
ACUAUGACAGCAGCCCUUU
2398
1584
AGGGCUGCUGUCAUAGUGA
2399
UCACUAUGACAGCAGCCCU
2400
1585
GGGCUGCUGUCAUAGUGAA
2401
UUCACUAUGACAGCAGCCC
2402
1587
GCUGCUGUCAUAGUGAACA
2403
UGUUCACUAUGACAGCAGC
2404
1589
UGCUGUCAUAGUGAACAAU
2405
AUUGUUCACUAUGACAGCA
2406
1594
UCAUAGUGAACAAUUUAUA
2407
UAUAAAUUGUUCACUAUGA
2408
1595
CAUAGUGAACAAUUUAUAA
2409
UUAUAAAUUGUUCACUAUG
2410
1610
AUAAGUGUCCCAUGGGGCA
2411
UGCCCCAUGGGACACUUAU
2412
1620
CAUGGGGCAGACACUCCUU
2413
AAGGAGUGUCUGCCCCAUG
2414
1621
AUGGGGCAGACACUCCUUU
2415
AAAGGAGUGUCUGCCCCAU
2416
1623
GGGGCAGACACUCCUUUUU
2417
AAAAAGGAGUGUCUGCCCC
2418
1624
GGGCAGACACUCCUUUUUU
2419
AAAAAAGGAGUGUCUGCCC
2420
1636
CUUUUUUCCCAGUCCUGCA
2421
UGCAGGACUGGGAAAAAAG
2422
1640
UUUCCCAGUCCUGCAACCU
2423
AGGUUGCAGGACUGGGAAA
2424
1645
CAGUCCUGCAACCUGGAUU
2425
AAUCCAGGUUGCAGGACUG
2426
1647
GUCCUGCAACCUGGAUUUU
2427
AAAAUCCAGGUUGCAGGAC
2428
1653
CAACCUGGAUUUUCUGCCU
2429
AGGCAGAAAAUCCAGGUUG
2430
1670
CUCAGCCCCAUUUUGCUGA
2431
UCAGCAAAAUGGGGCUGAG
2432
1694
AUGACUUUCUGAAUAAAGA
2433
UCUUUAUUCAGAAAGUCAU
2434
1695
UGACUUUCUGAAUAAAGAU
2435
AUCUUUAUUCAGAAAGUCA
2436
1704
GAAUAAAGAUGGCAACACA
2437
UGUGUUGCCAUCUUUAUUC
2438
1732
CCAUUUUCAGUUCUUACCU
2439
AGGUAAGAACUGAAAAUGG
2440
1736
UUUCAGUUCUUACCUGGGA
2441
UCCCAGGUAAGAACUGAAA
2442
1737
UUCAGUUCUUACCUGGGAA
2443
UUCCCAGGUAAGAACUGAA
2444
1741
GUUCUUACCUGGGAACCUA
2445
UAGGUUCCCAGGUAAGAAC
2446
1742
UUCUUACCUGGGAACCUAA
2447
UUAGGUUCCCAGGUAAGAA
2448
1743
UCUUACCUGGGAACCUAAU
2449
AUUAGGUUCCCAGGUAAGA
2450
1744
CUUACCUGGGAACCUAAUU
2451
AAUUAGGUUCCCAGGUAAG
2452
1749
CUGGGAACCUAAUUCCCCA
2453
UGGGGAAUUAGGUUCCCAG
2454
1751
GGGAACCUAAUUCCCCAGA
2455
UCUGGGGAAUUAGGUUCCC
2456
1752
GGAACCUAAUUCCCCAGAA
2457
UUCUGGGGAAUUAGGUUCC
2458
1757
CUAAUUCCCCAGAAGCUAA
2459
UUAGCUUCUGGGGAAUUAG
2460
1758
UAAUUCCCCAGAAGCUAAA
2461
UUUAGCUUCUGGGGAAUUA
2462
1759
AAUUCCCCAGAAGCUAAAA
2463
UUUUAGCUUCUGGGGAAUU
2464
1760
AUUCCCCAGAAGCUAAAAA
2465
UUUUUAGCUUCUGGGGAAU
2466
1763
CCCCAGAAGCUAAAAAACU
2467
AGUUUUUUAGCUUCUGGGG
2468
1764
CCCAGAAGCUAAAAAACUA
2469
UAGUUUUUUAGCUUCUGGG
2470
1776
AAAACUAGACAUUAGUUGU
2471
ACAACUAAUGUCUAGUUUU
2472
1777
AAACUAGACAUUAGUUGUU
2473
AACAACUAAUGUCUAGUUU
2474
1778
AACUAGACAUUAGUUGUUU
2475
AAACAACUAAUGUCUAGUU
2476
1779
ACUAGACAUUAGUUGUUUU
2477
AAAACAACUAAUGUCUAGU
2478
1782
AGACAUUAGUUGUUUUGGU
2479
ACCAAAACAACUAAUGUCU
2480
1783
GACAUUAGUUGUUUUGGUU
2481
AACCAAAACAACUAAUGUC
2482
1788
UAGUUGUUUUGGUUGCUUU
2483
AAAGCAACCAAAACAACUA
2484
1791
UUGUUUUGGUUGCUUUGUU
2485
AACAAAGCAACCAAAACAA
2486
1844
AUAUCCCUGGUAGUUUUGU
2487
ACAAAACUACCAGGGAUAU
2488
1847
UCCCUGGUAGUUUUGUGUU
2489
AACACAAAACUACCAGGGA
2490
1849
CCUGGUAGUUUUGUGUUAA
2491
UUAACACAAAACUACCAGG
2492
1854
UAGUUUUGUGUUAACCACU
2493
AGUGGUUAACACAAAACUA
2494
1861
GUGUUAACCACUGAUAACU
2495
AGUUAUCAGUGGUUAACAC
2496
1863
GUUAACCACUGAUAACUGU
2497
ACAGUUAUCAGUGGUUAAC
2498
1868
CCACUGAUAACUGUGGAAA
2499
UUUCCACAGUUAUCAGUGG
2500
1870
ACUGAUAACUGUGGAAAGA
2501
UCUUUCCACAGUUAUCAGU
2502
1882
GGAAAGAGCUAGGUCUACU
2503
AGUAGACCUAGCUCUUUCC
2504
1888
AGCUAGGUCUACUGAUAUA
2505
UAUAUCAGUAGACCUAGCU
2506
1890
CUAGGUCUACUGAUAUACA
2507
UGUAUAUCAGUAGACCUAG
2508
1893
GGUCUACUGAUAUACAAUA
2509
UAUUGUAUAUCAGUAGACC
2510
1894
GUCUACUGAUAUACAAUAA
2511
UUAUUGUAUAUCAGUAGAC
2512
1895
UCUACUGAUAUACAAUAAA
2513
UUUAUUGUAUAUCAGUAGA
2514
1897
UACUGAUAUACAAUAAACA
2515
UGUUUAUUGUAUAUCAGUA
2516
1905
UACAAUAAACAUGUGUGCA
2517
UGCACACAUGUUUAUUGUA
2518
1911
AAACAUGUGUGCAUCUUGA
2519
UCAAGAUGCACACAUGUUU
2520
1915
AUGUGUGCAUCUUGAACAA
2521
UUGUUCAAGAUGCACACAU
2522
1916
UGUGUGCAUCUUGAACAAU
2523
AUUGUUCAAGAUGCACACA
2524
1917
GUGUGCAUCUUGAACAAUU
2525
AAUUGUUCAAGAUGCACAC
2526
1922
CAUCUUGAACAAUUUGAGA
2527
UCUCAAAUUGUUCAAGAUG
2528
1927
UGAACAAUUUGAGAGGGGA
2529
UCCCCUCUCAAAUUGUUCA
2530
1930
ACAAUUUGAGAGGGGAGGU
2531
ACCUCCCCUCUCAAAUUGU
2532
1954
UGGAAAUGUGGGUGUUCCU
2533
AGGAACACCCACAUUUCCA
2534
1958
AAUGUGGGUGUUCCUGUUU
2535
AAACAGGAACACCCACAUU
2536
1962
UGGGUGUUCCUGUUUUUUU
2537
AAAAAAACAGGAACACCCA
2538
2007
UUAAUGAGCUCACCCUUUA
2539
UAAAGGGUGAGCUCAUUAA
2540
2008
UAAUGAGCUCACCCUUUAA
2541
UUAAAGGGUGAGCUCAUUA
2542
2010
AUGAGCUCACCCUUUAACA
2543
UGUUAAAGGGUGAGCUCAU
2544
2012
GAGCUCACCCUUUAACACA
2545
UGUGUUAAAGGGUGAGCUC
2546
2014
GCUCACCCUUUAACACAAA
2547
UUUGUGUUAAAGGGUGAGC
2548
2016
UCACCCUUUAACACAAAAA
2549
UUUUUGUGUUAAAGGGUGA
2550
2017
CACCCUUUAACACAAAAAA
2551
UUUUUUGUGUUAAAGGGUG
2552
2021
CUUUAACACAAAAAAAGCA
2553
UGCUUUUUUUGUGUUAAAG
2554
2028
ACAAAAAAAGCAAGGUGAU
2555
AUCACCUUGCUUUUUUUGU
2556
2044
GAUGUAUUUUAAAAAAGGA
2557
UCCUUUUUUAAAAUACAUC
2558
2060
GGAAGUGGAAAUAAAAAAA
2559
UUUUUUUAUUUCCACUUCC
2560
2072
AAAAAAAUCUCAAAGCUAU
2561
AUAGCUUUGAGAUUUUUUU
2562
2073
AAAAAAUCUCAAAGCUAUU
2563
AAUAGCUUUGAGAUUUUUU
2564
2081
UCAAAGCUAUUUGAGUUCU
2565
AGAACUCAAAUAGCUUUGA
2566
2084
AAGCUAUUUGAGUUCUCGU
2567
ACGAGAACUCAAAUAGCUU
2568
2086
GCUAUUUGAGUUCUCGUCU
2569
AGACGAGAACUCAAAUAGC
2570
2098
CUCGUCUGUCCCUAGCAGU
2571
ACUGCUAGGGACAGACGAG
2572
2100
CGUCUGUCCCUAGCAGUCU
2573
AGACUGCUAGGGACAGACG
2574
2105
GUCCCUAGCAGUCUUUCUU
2575
AAGAAAGACUGCUAGGGAC
2576
2121
CUUCAGCUCACUUGGCUCU
2577
AGAGCCAAGUGAGCUGAAG
2578
2123
UCAGCUCACUUGGCUCUCU
2579
AGAGAGCCAAGUGAGCUGA
2580
2124
CAGCUCACUUGGCUCUCUA
2581
UAGAGAGCCAAGUGAGCUG
2582
2132
UUGGCUCUCUAGAUCCACU
2583
AGUGGAUCUAGAGAGCCAA
2584
2134
GGCUCUCUAGAUCCACUGU
2585
ACAGUGGAUCUAGAGAGCC
2586
2137
UCUCUAGAUCCACUGUGGU
2587
ACCACAGUGGAUCUAGAGA
2588
2142
AGAUCCACUGUGGUUGGCA
2589
UGCCAACCACAGUGGAUCU
2590
2144
AUCCACUGUGGUUGGCAGU
2591
ACUGCCAACCACAGUGGAU
2592
2145
UCCACUGUGGUUGGCAGUA
2593
UACUGCCAACCACAGUGGA
2594
2146
CCACUGUGGUUGGCAGUAU
2595
AUACUGCCAACCACAGUGG
2596
2155
UUGGCAGUAUGACCAGAAU
2597
AUUCUGGUCAUACUGCCAA
2598
2157
GGCAGUAUGACCAGAAUCA
2599
UGAUUCUGGUCAUACUGCC
2600
2161
GUAUGACCAGAAUCAUGGA
2601
UCCAUGAUUCUGGUCAUAC
2602
2171
AAUCAUGGAAUUUGCUAGA
2603
UCUAGCAAAUUCCAUGAUU
2604
2172
AUCAUGGAAUUUGCUAGAA
2605
UUCUAGCAAAUUCCAUGAU
2606
2176
UGGAAUUUGCUAGAACUGU
2607
ACAGUUCUAGCAAAUUCCA
2608
2180
AUUUGCUAGAACUGUGGAA
2609
UUCCACAGUUCUAGCAAAU
2610
2184
GCUAGAACUGUGGAAGCUU
2611
AAGCUUCCACAGUUCUAGC
2612
2198
AGCUUCUACUCCUGCAGUA
2613
UACUGCAGGAGUAGAAGCU
2614
2199
GCUUCUACUCCUGCAGUAA
2615
UUACUGCAGGAGUAGAAGC
2616
2206
CUCCUGCAGUAAGCACAGA
2617
UCUGUGCUUACUGCAGGAG
2618
2217
AGCACAGAUCGCACUGCCU
2619
AGGCAGUGCGAUCUGUGCU
2620
2220
ACAGAUCGCACUGCCUCAA
2621
UUGAGGCAGUGCGAUCUGU
2622
2221
CAGAUCGCACUGCCUCAAU
2623
AUUGAGGCAGUGCGAUCUG
2624
2222
AGAUCGCACUGCCUCAAUA
2625
UAUUGAGGCAGUGCGAUCU
2626
2223
GAUCGCACUGCCUCAAUAA
2627
UUAUUGAGGCAGUGCGAUC
2628
2229
ACUGCCUCAAUAACUUGGU
2629
ACCAAGUUAUUGAGGCAGU
2630
2231
UGCCUCAAUAACUUGGUAU
2631
AUACCAAGUUAUUGAGGCA
2632
2237
AAUAACUUGGUAUUGAGCA
2633
UGCUCAAUACCAAGUUAUU
2634
2240
AACUUGGUAUUGAGCACGU
2635
ACGUGCUCAAUACCAAGUU
2636
2243
UUGGUAUUGAGCACGUAUU
2637
AAUACGUGCUCAAUACCAA
2638
2255
ACGUAUUUUGCAAAAGCUA
2639
UAGCUUUUGCAAAAUACGU
2640
2257
GUAUUUUGCAAAAGCUACU
2641
AGUAGCUUUUGCAAAAUAC
2642
2258
UAUUUUGCAAAAGCUACUU
2643
AAGUAGCUUUUGCAAAAUA
2644
2259
AUUUUGCAAAAGCUACUUU
2645
AAAGUAGCUUUUGCAAAAU
2646
2268
AAGCUACUUUUCCUAGUUU
2647
AAACUAGGAAAAGUAGCUU
2648
2271
CUACUUUUCCUAGUUUUCA
2649
UGAAAACUAGGAAAAGUAG
2650
2279
CCUAGUUUUCAGUAUUACU
2651
AGUAAUACUGAAAACUAGG
2652
2280
CUAGUUUUCAGUAUUACUU
2653
AAGUAAUACUGAAAACUAG
2654
2312
AUCCCUUUAAUUUCUUGCU
2655
AGCAAGAAAUUAAAGGGAU
2656
2326
UUGCUUGAAAAUCCCAUGA
2657
UCAUGGGAUUUUCAAGCAA
2658
2327
UGCUUGAAAAUCCCAUGAA
2659
UUCAUGGGAUUUUCAAGCA
2660
2329
CUUGAAAAUCCCAUGAACA
2661
UGUUCAUGGGAUUUUCAAG
2662
2343
GAACAUUAAAGAGCCAGAA
2663
UUCUGGCUCUUUAAUGUUC
2664
2346
CAUUAAAGAGCCAGAAAUA
2665
UAUUUCUGGCUCUUUAAUG
2666
2355
GCCAGAAAUAUUUUCCUUU
2667
AAAGGAAAAUAUUUCUGGC
2668
2367
UUCCUUUGUUAUGUACGGA
2669
UCCGUACAUAACAAAGGAA
2670
2368
UCCUUUGUUAUGUACGGAU
2671
AUCCGUACAUAACAAAGGA
2672
2369
CCUUUGUUAUGUACGGAUA
2673
UAUCCGUACAUAACAAAGG
2674
2370
CUUUGUUAUGUACGGAUAU
2675
AUAUCCGUACAUAACAAAG
2676
2371
UUUGUUAUGUACGGAUAUA
2677
UAUAUCCGUACAUAACAAA
2678
2372
UUGUUAUGUACGGAUAUAU
2679
AUAUAUCCGUACAUAACAA
2680
2373
UGUUAUGUACGGAUAUAUA
2681
UAUAUAUCCGUACAUAACA
2682
2394
UAUAUAGUCUUCCAAGAUA
2683
UAUCUUGGAAGACUAUAUA
2684
2401
UCUUCCAAGAUAGAAGUUU
2685
AAACUUCUAUCUUGGAAGA
2686
2404
UCCAAGAUAGAAGUUUACU
2687
AGUAAACUUCUAUCUUGGA
2688
2405
CCAAGAUAGAAGUUUACUU
2689
AAGUAAACUUCUAUCUUGG
2690
2448
UUCCAGAUAAGACAUGUCA
2691
UGACAUGUCUUAUCUGGAA
2692
2454
AUAAGACAUGUCACCAUUA
2693
UAAUGGUGACAUGUCUUAU
2694
2456
AAGACAUGUCACCAUUAAU
2695
AUUAAUGGUGACAUGUCUU
2696
2459
ACAUGUCACCAUUAAUUCU
2697
AGAAUUAAUGGUGACAUGU
2698
2461
AUGUCACCAUUAAUUCUCA
2699
UGAGAAUUAAUGGUGACAU
2700
2462
UGUCACCAUUAAUUCUCAA
2701
UUGAGAAUUAAUGGUGACA
2702
2465
CACCAUUAAUUCUCAACGA
2703
UCGUUGAGAAUUAAUGGUG
2704
2467
CCAUUAAUUCUCAACGACU
2705
AGUCGUUGAGAAUUAAUGG
2706
2470
UUAAUUCUCAACGACUGCU
2707
AGCAGUCGUUGAGAAUUAA
2708
2472
AAUUCUCAACGACUGCUCU
2709
AGAGCAGUCGUUGAGAAUU
2710
2474
UUCUCAACGACUGCUCUAU
2711
AUAGAGCAGUCGUUGAGAA
2712
2475
UCUCAACGACUGCUCUAUU
2713
AAUAGAGCAGUCGUUGAGA
2714
2476
CUCAACGACUGCUCUAUUU
2715
AAAUAGAGCAGUCGUUGAG
2716
2479
AACGACUGCUCUAUUUUGU
2717
ACAAAAUAGAGCAGUCGUU
2718
2488
UCUAUUUUGUUGUACGGUA
2719
UACCGUACAACAAAAUAGA
2720
2490
UAUUUUGUUGUACGGUAAU
2721
AUUACCGUACAACAAAAUA
2722
2491
AUUUUGUUGUACGGUAAUA
2723
UAUUACCGUACAACAAAAU
2724
2493
UUUGUUGUACGGUAAUAGU
2725
ACUAUUACCGUACAACAAA
2726
2494
UUGUUGUACGGUAAUAGUU
2727
AACUAUUACCGUACAACAA
2728
2495
UGUUGUACGGUAAUAGUUA
2729
UAACUAUUACCGUACAACA
2730
2496
GUUGUACGGUAAUAGUUAU
2731
AUAACUAUUACCGUACAAC
2732
2501
ACGGUAAUAGUUAUCACCU
2733
AGGUGAUAACUAUUACCGU
2734
2506
AAUAGUUAUCACCUUCUAA
2735
UUAGAAGGUGAUAACUAUU
2736
2507
AUAGUUAUCACCUUCUAAA
2737
UUUAGAAGGUGAUAACUAU
2738
2521
CUAAAUUACUAUGUAAUUU
2739
AAAUUACAUAGUAAUUUAG
2740
2543
CACUUAUUAUGUUUAUUGU
2741
ACAAUAAACAUAAUAAGUG
2742
2555
UUAUUGUCUUGUAUCCUUU
2743
AAAGGAUACAAGACAAUAA
2744
2564
UGUAUCCUUUCUCUGGAGU
2745
ACUCCAGAGAAAGGAUACA
2746
2566
UAUCCUUUCUCUGGAGUGU
2747
ACACUCCAGAGAAAGGAUA
2748
2571
UUUCUCUGGAGUGUAAGCA
2749
UGCUUACACUCCAGAGAAA
2750
2574
CUCUGGAGUGUAAGCACAA
2751
UUGUGCUUACACUCCAGAG
2752
2575
UCUGGAGUGUAAGCACAAU
2753
AUUGUGCUUACACUCCAGA
2754
2580
AGUGUAAGCACAAUGAAGA
2755
UCUUCAUUGUGCUUACACU
2756
2586
AGCACAAUGAAGACAGGAA
2757
UUCCUGUCUUCAUUGUGCU
2758
2588
CACAAUGAAGACAGGAAUU
2759
AAUUCCUGUCUUCAUUGUG
2760
2589
ACAAUGAAGACAGGAAUUU
2761
AAAUUCCUGUCUUCAUUGU
2762
2594
GAAGACAGGAAUUUUGUAU
2763
AUACAAAAUUCCUGUCUUC
2764
2613
AUUUUUAACCAAUGCAACA
2765
UGUUGCAUUGGUUAAAAAU
2766
2619
AACCAAUGCAACAUACUCU
2767
AGAGUAUGUUGCAUUGGUU
2768
2624
AUGCAACAUACUCUCAGCA
2769
UGCUGAGAGUAUGUUGCAU
2770
2627
CAACAUACUCUCAGCACCU
2771
AGGUGCUGAGAGUAUGUUG
2772
2628
AACAUACUCUCAGCACCUA
2773
UAGGUGCUGAGAGUAUGUU
2774
2629
ACAUACUCUCAGCACCUAA
2775
UUAGGUGCUGAGAGUAUGU
2776
2630
CAUACUCUCAGCACCUAAA
2777
UUUAGGUGCUGAGAGUAUG
2778
2646
AAAAUAGUGCCGGGAACAU
2779
AUGUUCCCGGCACUAUUUU
2780
2649
AUAGUGCCGGGAACAUAGU
2781
ACUAUGUUCCCGGCACUAU
2782
2656
CGGGAACAUAGUAAGGGCU
2783
AGCCCUUACUAUGUUCCCG
2784
2660
AACAUAGUAAGGGCUCAGU
2785
ACUGAGCCCUUACUAUGUU
2786
2667
UAAGGGCUCAGUAAAUACU
2787
AGUAUUUACUGAGCCCUUA
2788
2668
AAGGGCUCAGUAAAUACUU
2789
AAGUAUUUACUGAGCCCUU
2790
2682
UACUUGUUGAAUAAACUCA
2791
UGAGUUUAUUCAACAAGUA
2792
2684
CUUGUUGAAUAAACUCAGU
2793
ACUGAGUUUAUUCAACAAG
2794
2698
UCAGUCUCCUACAUUAGCA
2795
UGCUAAUGUAGGAGACUGA
2796
2700
AGUCUCCUACAUUAGCAUU
2797
AAUGCUAAUGUAGGAGACU
2798
2702
UCUCCUACAUUAGCAUUCU
2799
AGAAUGCUAAUGUAGGAGA
2800
2703
CUCCUACAUUAGCAUUCUA
2801
UAGAAUGCUAAUGUAGGAG
2802
2704
UCCUACAUUAGCAUUCUAA
2803
UUAGAAUGCUAAUGUAGGA
2804
Example 8
In Vivo Dose Response of EGLN Cocktail in Liver
In order to evaluate the efficacy of the iRNA agents directed to EGLN genes, dose response studies were conducted targeting individual EGLN genes and combinations of EGLN genes in the liver. For these studies, mice (3 animals per group) were injected IV with formulations at the doses outlined in Table 7. A mix of EGLN1 and EGLN3, EGLN1 and EGLN2, EGLN2 and EGLN3 and EGLN1, EGLN2 and EGLN3 formulations were tested to confirm if co-injection of individual LNP11 formulations with siRNA against single targets worked as well as injection of a single formulation with siRNAs against all 3 EGLN targets. At 6 days after the second dose the animals were sacrificed and the livers were evaluated for bDNA. Serum was evaluated for EPO measurements by ELISA. The results are shown in FIG. 11.
It was found that each EGLN specific siRNA produced specific and robust knockdown in the liver. Furthermore, synergies were detected when the siRNA to more than one EGLN targeting siRNA was used.
TABLE 7
In vivo knockdown of EGLN genes
Dose
Group
siRNA
(mg/kg)
PBS
—
Luciferase
AD-1955
0.5
EGLN1
AD-40894
0.5
EGLN2
AD-40773
0.5
EGLN3
AD-40758
0.5
EGLN1 + 2
AD-40894 (50%)
0.5/0.5
AD-40773 (50%)
EGLN1 + 3
AD-40894 (50%)
0.5/0.5
AD-40758 (50%)
EGLN2 + 3
AD-40773 (50%)
0.5/0.5
AD-40758 (50%)
EGLN1 + 2 + 3
AD-40894 (33%)
0.5/0.5/0.5
AD-40773 (33%)
AD-40758 (33%)
Example 9
In Vivo Production of Erythropoietin and Hematology Using EGLN Cocktail
In order to determine whether the administration of an EGLN iRNA cocktail was capable of increasing erythropoietin expression in vivo, a study was designed according to Table 8. Female C57B6 mice were dosed IV with PBS or LNP11-1955 luciferase controls, three different EGLN siRNA formulations or four different mixes of EGLN siRNA formulations. At day 6, a second dose was administered. On day 12, plasma samples were taken, animals were sacrificed and livers were harvested for measurement of EGLN1, EGLN2, EGLN3 and EPO mRNA. Also on day 12 blood was drawn (hematogology measurements including a count of reticulocytes, red blood cells, hemoglobin measurements and hematocrit levels) and animals were sacrified and the livers were taken for bDNA analysis. The data are shown in FIGS. 12 and 13.
TABLE 8
In vivo knockdown of EGLN genes
Dose
Group
siRNA
(mg/kg)
PBS
—
Luciferase
AD-1955
0.5
EGLN1
AD-40894
0.5
EGLN2
AD-40773
0.5
EGLN3
AD-40758
0.5
EGLN1 + 2
AD-40894 (50%)
0.5/0.5
AD-40773 (50%)
EGLN1 + 3
AD-40894 (50%)
0.5/0.5
AD-40758 (50%)
EGLN2 + 3
AD-40773 (50%)
0.5/0.5
AD-40758 (50%)
EGLN1 + 2 + 3
AD-40894 (33%)
0.5/0.5/0.5
AD-40773 (33%)
AD-40758 (33%)
It can be seen from FIGS. 12 and 13 that targeting EGLN1 alone or in combination with other EGLN genes increases serum EPO levels. It is suggested herein that knockdown of EGLN1 and/or EGLN2 induces feedback loop up-regulation of EGLN3 mimicking hypoxic response.
In general, a considerable increase in reticulocytes versus control was obsevered with an even larger increase in hematocrit, RBC count and hemoglobin levels in the plasma. Therefore, it has been surprisingly discovered that knockdown of EGLN1 (either alone or in combination) which produced an increase in EPO, concomitantly stimulated erythropiesis.
Example 10
Downregulation of Hepcidin
In order to evaluate the efficacy of the iRNA agents on the downregulation of Hepcidin dose response studies were conducted for iRNAs targeting individual EGLN genes and combinations of EGLNs in the liver. For these studies, mice (5 animals per group) were injected IV with formulations at the doses outlined in Table 9. Animals were dosed at day 1 and day 6. At day 12, the animals were bled and sacrificed and the livers were taken. The levels of hepcidin in liver were measured by bDNA. Downregulation of Hepcidin was observed when the formulations included at least EGLN1 (alone or in combination). The results are shown in FIG. 14.
TABLE 9
Downregulation of Hepcidin
Dose
Group
siRNA
(mg/kg)
PBS
—
Luciferase
AD-1955
0.5
EGLN1
AD-40894
0.5
EGLN2
AD-40773
0.5
EGLN3
AD-40758
0.5
EGLN1 + 2
AD-40894 (50%)
0.5/0.5
AD-40773 (50%)
EGLN1 + 3
AD-40894 (50%)
0.5/0.5
AD-40758 (50%)
EGLN2 + 3
AD-40773 (50%)
0.5/0.5
AD-40758 (50%)
EGLN1 + 2 + 3
AD-40894 (33%)
0.5/0.5/0.5
AD-40773 (33%)
AD-40758 (33%)
Example 11
Tissue Specificity
In order to determine whether administration of an EGLN iRNA cocktail was capable of tissue specificity, a study was designed according to Table 10. Female C57B6 mice were dosed four times, at day 1, 8, 15 and 22, by IV with LNP11-1955 luciferase control or a cocktail of EGLN siRNA formulation. On day 29, a set of plasma samples were taken, animals were sacrificed and livers, kidneys and spleens were harvested for measurement of EGLN1, EGLN2, EGLN3 and EPO mRNA measurements again by branched DNA analysis.
TABLE 10
Tissue specificity
Dose
Group
siRNA
(mg/kg)
Luciferase
AD-1955
EGLN mix
AD-40894 (.375 mg/kg)
1.5 total
AD-40773 (.75 mg/kg)
AD-40758 (.375 mg/kg)
It can be seen from FIG. 15 that the EGLN cocktail stimulated EPO in the liver and showed little to no stimulation in the kidneys and spleen. Hence the increase in serum EPO must arise from the liver. Liver tissue was removed and stained with oil red oil and H&E and compared to the positive control for fatty liver. Tissue staining revealed that weekly dosing (up to one month) was well tolerated by the liver.
Example 12
Durable Effects of Cocktail Administration
In order to determine durability of administration of an EGLN iRNA cocktail on the regulation of EPO and hematocrit, a study was designed according to Table 11. Female C57B6 mice were dosed IV with LNP11-1955 luciferase control or a formulation of a mix of EGLN siRNA as outlined in Table 11. Two groups of mice were dosed at either (1) only day 1 or (2) on days 1 and 6. At days 6, 11, 16, and 22 serum EPO was measured. At days 6, 11, 16, and 22, 27 and 33 hematocrit was measured. The results are shown in FIG. 16.
TABLE 11
Durable effects of cocktail administration
on Epo and hematocrit
Dose
Group
siRNA
(mg/kg)
Luciferase
AD-1955
1.5
EGLN mix
AD-40894 (.5 mg/kg)
1.5
(day 1 dose)
AD-40773 (.5 mg/kg)
AD-40758 (.5 mg/kg)
EGLN mix
AD-40894 (.5 mg/kg)
1.5
(day 1 and 6
AD-40773 (.5 mg/kg)
dose)
AD-40758 (.5 mg/kg)
It can be seen from FIG. 16 that the knockdown by the EGLN mix was sustained over a prolonged period of time. The durability of a single dose could be seen in the samples taken for hematocrit showing lasting effects of over one month. Prolonged effects of the administration of the EGLN cocktail were also seen in the increased levels of EPO which lasted about 2 weeks after a single dose of the cocktail.
Example 13
Studies in an Animal Model of Anemia
Studies of the effects of the iRNA agents (alone or in combination) on a mouse model of anemia were performed to evaluate therapeutic outcomes and efficacy. Endpoints included target knockdown of each of the EGLN genes as well as hepcidin, improved EPO production, hematology measurements (including red blood cell count, Hemoglobin levels, hematocrit levels, and reticulocyte levels), and iron parameters (including serum iron level, transferrin saturation (% TSAT), unsaturated iron binding capacity (UIBC), total iron binding capacity (TIBC) and ferritin levels).
FBVN mice which had undergone 5/6 nephrectomy (Charles River Laboratories; Wilmington, Mass.) were dosed three times, at day 0, 4 and 8. Dosing involved IV administration at 1 mg/kg of the siRNA or siRNAs outlined in Table 12 formulated in LNP11. The study also included control groups of SHAM and PBS treated control groups as well as a control group containing the Luciferase siRNA AD-1955. At day 12 the animals were sacrificed, with terminal bleeds made and tissues removed for mRNA analysis. In all cases, the levels are normalized to levels of actin and presented as a percent sham. The results are presented in FIGS. 17-22 and discussed below.
TABLE 12
In vivo studies in a model of anemia
Sample
Dose
Group
siRNA
Formulation
Size (n)
(mg/kg)
SHAM
—
5
PBS
—
5
Luciferase
AD-1955
LNP11
5
1
(control)
EGLN1
AD-40894
LNP11
4
1
EGLN1-2
AD-40894 (50%)
LNP11
5
1 (0.5 ea)
AD-40773 (50%)
EGLN1-2-3
AD-40894 (33%)
LNP11
4
1 (0.33 ea)
mix
AD-40773 (33%)
AD-40758 (33%)
Target mRNA Knockdown (EGLN and Hepcidin)
Results of measurement of EGLN 1, 2, and 3 in liver as well as hepcidin expression is shown in FIGS. 17 and 18, respectively. It can be seen from the data that, just as with previous studies, the effects of the iRNA agents targeting the EGLN genes, either alone or in combination are specific and robust. There was upregulation of EGLN3 mRNA seen previously due to feedback regulation particularly in EGLN1-2 treated groups.
Downregulation of hepcidin (HAMP1) was observed when the formulations included at least EGLN1 (alone or in combination). Clearly, knockdown of EGLN1, EGLN1-2 and EGLN1-2-3 was shown to induce a down regulation of Hepcidin mRNA in the liver.
Improved EPO Production
Measurements of erythropoietin were made at the terminal bleed at day 12 and the data are shown in FIG. 19. It can be seen that knockdown of EGLN1-2 and EGLN1-2-3 significantly increased liver EPO mRNA in the context of 5/6 nephrectomy. An increase in EPO mRNA was not detected with EGLN1 knockdown consistent with previous experiments where the increase was only seen at the protein level. These results suggest that in anemic patients, administration of the iRNA agents targeting EGLN genes may serve a therapeutic need to increase EPO.
Hematology
Hematocrit levels of the test groups were measured at day 0 and at sacrifice on day 12. The pre and post values of the animals are shown in FIG. 20. As can be seen from the data, there was a significant increase in Hematocrit in double and triple combo groups with a more minor effect seen in EGLN1 alone treated animals compared to SHAM controls.
Measurements of red blood cell count, Hemoglobin, and reticulocyte levels were also made at day 12 and good increases in hemoglobin and reticulocytes in all EGLN groups was observed. See FIG. 21.
Iron Parameters
Parameters associated with the etiology of anemia and erythropoiesis were also measured at day 12. These data are presented in FIG. 22. Decreases seen in TSAT, and increases in UIBC and TIBC in the double and triple combo EGLN knockdown animals was very informative. These data suggest that there might not be sufficient iron available to meet the enhanced erythropoiesis demand (due to stimulation by the iRNA agents administered) of the system. In other words, the effect of the iRNA agents in enhancing erythropoiesis was so successful, it outpaced (or drained) the iron pool of the animal. These data suggest that the iRNA agents may be even more effective if administered in conjunction with an iron supplement.
Example 14
Design and Synthesis of siRNA Targeting Human EGLN Genes
Oligonucleotide design was carried out to identify siRNAs targeting the genes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. The design process used the EGLN transcript NM—022051.2 for EGLN1 (SEQ ID NO: 390), NM—053046.2 for EGLN2 (SEQ ID NO: 391), and NM—022073.3 for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBI Refseq collection. All siRNA duplexes were designed that shared 100% identity with the listed human and rhesus transcripts. The constructs are shown in Tables 13A, B and C.
TABLE 13A
Human EGNL1 Single Strands and Duplex Sequences
For all the sequences in the list, ‘endolight’ chemistry was
applied as described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Sense
NO
Antisense
NO
AD-
cAcGAcAccGGGAAGuucAdTsdT
2807
UGAACUUCCCGGUGUCGUGdTsdT
2808
47677.1
AD-
GAcuGGGAuGccAAGGuAAdTsdT
2809
UuACCUUGGcAUCCcAGUCdTsdT
2810
47683.1
AD-
ccAAGGuAAGuGGAGGuAudTsdT
2811
AuACCUCcACUuACCUUGGdTsdT
2812
47688.1
AD-
GuGGAGGuAuAcuucGAAudTsdT
2813
AUUCGAAGuAuACCUCcACdTsdT
2814
47694.1
AD-
GuGGAGGuAuAcuucGAAudTsdT
2815
AUUCGAAGuAuACCUCcACdTsdT
2816
47694.2
AD-
GAGGuAuAcuucGAAuuuudTsdT
2817
AAAAUUCGAAGuAuACCUCdTsdT
2818
47700.1
AD-
ccAAAuuuGAuAGAcuGcudTsdT
2819
AGcAGUCuAUcAAAUUUGGdTsdT
2820
47706.1
AD-
GcuAcAAGGuAcGcAAuAAdTsdT
2821
UuAUUGCGuACCUUGuAGCdTsdT
2822
47711.1
AD-
GAGAGcAcGAGcuAAAGuAdTsdT
2823
uACUUuAGCUCGUGCUCUCdTsdT
2824
47716.1
AD-
GAGcuAAAGuAAAAuAucudTsdT
2825
AGAuAUUUuACUUuAGCUCdTsdT
2826
47678.1
AD-
GuGuGAGGGuuGAAcucAAdTsdT
2827
UUGAGUUcAACCCUcAcACdTsdT
2828
47689.1
AD-
GuGAGGGuuGAAcucAAuAdTsdT
2829
uAUUGAGUUcAACCCUcACdTsdT
2830
47695.1
AD-
GGuuGAAcucAAuAAAccudTsdT
2831
AGGUUuAUUGAGUUcAACCdTsdT
2832
47701.1
AD-
GAcGucuucuAGAGccuuudTsdT
2833
AAAGGCUCuAGAAGACGUCdTsdT
2834
47707.1
AD-
ccAGAucuGuuAucuAGcudTsdT
2835
AGCuAGAuAAcAGAUCUGGdTsdT
2836
47712.1
AD-
GuuAucuAGcuGAGuucAudTsdT
2837
AUGAACUcAGCuAGAuAACdTsdT
2838
47717.1
AD-
GGuAcAAuuuAucuAAAcudTsdT
2839
AGUUuAGAuAAAUUGuACCdTsdT
2840
47679.1
AD-
ccucuuAAuAAuGAuuGuudTsdT
2841
AAcAAUcAUuAUuAAGAGGdTsdT
2842
47684.1
AD-
GccAGuGAcuGAuGAuuAAdTsdT
2843
UuAAUcAUcAGUcACUGGCdTsdT
2844
47690.1
AD-
ccAGuGAcuGAuGAuuAAudTsdT
2845
AUuAAUcAUcAGUcACUGGdTsdT
2846
47696.1
AD-
GAGcAcuuuAAuuAcAAcudTsdT
2847
AGUUGuAAUuAAAGUGCUCdTsdT
2848
47702.1
AD-
ccAuuuAcuAccAAuAAcudTsdT
2849
AGUuAUUGGuAGuAAAUGGdTsdT
2850
47708.1
AD-
GGcuGGGGuuuAAGuuAAAdTsdT
2851
UUuAACUuAAACCCcAGCCdTsdT
2852
47713.1
AD-
GcuGGGGuuuAAGuuAAAudTsdT
2853
AUUuAACUuAAACCCcAGCdTsdT
2854
47718.1
AD-
cuucAAGuuccuAAGAuAAdTsdT
2855
UuAUCUuAGGAACUUGAAGdTsdT
2856
47680.1
AD-
GGGcuuucuuAAGcuuucAdTsdT
2857
UGAAAGCUuAAGAAAGCCCdTsdT
2858
47685.1
AD-
cuuAGAcuucAcuuuccuAdTsdT
2859
uAGGAAAGUGAAGUCuAAGdTsdT
2860
47691.1
AD-
cuucAcuuuccuAGGcuuudTsdT
2861
AAAGCCuAGGAAAGUGAAGdTsdT
2862
47697.1
AD-
cuAucucuGuccuuGAucudTsdT
2863
AGAUcAAGGAcAGAGAuAGdTsdT
2864
47703.1
AD-
GccAAAAuGuGAGuAuAcAdTsdT
2865
UGuAuACUcAcAUUUUGGCdTsdT
2866
47709.1
AD-
cAAAAuGuGAGuAuAcAGAdTsdT
2867
UCUGuAuACUcAcAUUUUGdTsdT
2868
47714.1
AD-
cuuAGGAGAAuuuGcAGGAdTsdT
2869
UCCUGcAAAUUCUCCuAAGdTsdT
2870
47719.1
AD-
GcGuuAGGccAcAAcucAAdTsdT
2871
UUGAGUUGUGGCCuAACGCdTsdT
2872
47686.1
AD-
cGuuAGGccAcAAcucAAAdTsdT
2873
UUUGAGUUGUGGCCuAACGdTsdT
2874
47692.1
AD-
cuAucuGuGGGuuGuGcuudTsdT
2875
AAGcAcAACCcAcAGAuAGdTsdT
2876
47698.1
AD-
cAGAcAGGucuuAAAuuGudTsdT
2877
AcAAUUuAAGACCUGUCUGdTsdT
2878
47704.1
AD-
GGAAAAGuuuAuAuAcucudTsdT
2879
AGAGuAuAuAAACUUUUCCdTsdT
2880
47710.1
AD-
cuGuuuGuGGccuAuAuGudTsdT
2881
AcAuAuAGGCcAcAAAcAGdTsdT
2882
47715.1
AD-
GuuuGuGGccuAuAuGuGudTsdT
2883
AcAcAuAuAGGCcAcAAACdTsdT
2884
47720.1
AD-
GuGuuuAAuccuGGuuAAAdTsdT
2885
UUuAACcAGGAUuAAAcACdTsdT
2886
47682.1
AD-
GuuuAAuccuGGuuAAAGAdTsdT
2887
UCUUuAACcAGGAUuAAACdTsdT
2888
47687.1
AD-
cuGuuuuuAuucAAcAcAudTsdT
2889
AUGUGUUGAAuAAAAAcAGdTsdT
2890
47693.1
AD-
cAuAuAcAGAuAGAcuAuAdTsdT
2891
uAuAGUCuAUCUGuAuAUGdTsdT
2892
47699.1
AD-
cAAGuuGcuuGuAAAGcuAdTsdT
2893
uAGCUUuAcAAGcAACUUGdTsdT
2894
47705.1
AD-
GcuuGuAAAGcuAAucuAAdTsdT
2895
UuAGAUuAGCUUuAcAAGCdTsdT
2896
40932.2
AD-
GcuuGuAAAGcuAAucuAAdTsdT
2897
UuAGAUuAGCUUuAcAAGCdTsdT
2898
40932.1
AD-
GcuuGuAAAGcuAAucuAAdTsdT
2899
UuAGAUuAGCUUuAcAAGCdTsdT
2900
40932.3
TABLE 13B
Human EGNL2 Single Strands and Duplex Sequences
For all the sequences in the list, ‘endolight’ chemistry
was applied as described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Sense
NO
Antisense
NO
AD-
cuucccAAGcccuuAGGGAdTsdT
2901
UCCCuAAGGGCUUGGGAAGdTsdT
2902
47721.1
AD-
cuuGGGGAccAGcAAGcAAdTsdT
2903
UUGCUUGCUGGUCCCcAAGdTsdT
2904
47727.1
AD-
cAuGcccGGGGGAuGAAGAdTsdT
2905
UCUUcAUCCCCCGGGcAUGdTsdT
2906
47733.1
AD-
cccGGGGGAuGAAGAcAcudTsdT
2907
AGUGUCUUcAUCCCCCGGGdTsdT
2908
47738.1
AD-
GGGGGAuGAAGAcAcuGcudTsdT
2909
AGcAGUGUCUUcAUCCCCCdTsdT
2910
47744.1
AD-
GcAGccccuAAGucAGGcudTsdT
2911
AGCCUGACUuAGGGGCUGCdTsdT
2912
47750.1
AD-
cAGuuAccAGGGucuucGudTsdT
2913
ACGAAGACCCUGGuAACUGdTsdT
2914
47756.1
AD-
GAGGcccccAAAcGGAAAudTsdT
2915
AUUUCCGUUUGGGGGCCUCdTsdT
2916
47722.1
AD-
GGGccAGGcAAGAGAAccAdTsdT
2917
UGGUUCUCUUGCCUGGCCCdTsdT
2918
47728.1
AD-
GccuGGcccuGGAcuAuAudTsdT
2919
AuAuAGUCcAGGGCcAGGCdTsdT
2920
47734.1
AD-
GcAuGcGGuAcuAcGGcAudTsdT
2921
AUGCCGuAGuACCGcAUGCdTsdT
2922
47739.1
AD-
GGuAcuAcGGcAucuGcGudTsdT
2923
ACGcAGAUGCCGuAGuACCdTsdT
2924
47745.1
AD-
cAuccGuGGGGAccAGAuudTsdT
2925
AAUCUGGUCCCcACGGAUGdTsdT
2926
47751.1
AD-
cGGGuAcGuAAGGcAcGuudTsdT
2927
AACGUGCCUuACGuACCCGdTsdT
2928
47763.1
AD-
GGuAcGuAAGGcAcGuuGAdTsdT
2929
UcAACGUGCCUuACGuACCdTsdT
2930
47723.1
AD-
cGcuGcAucAccuGuAucudTsdT
2931
AGAuAcAGGUGAUGcAGCGdTsdT
2932
47729.1
AD-
GcAucAccuGuAucuAuuAdTsdT
2933
uAAuAGAuAcAGGUGAUGCdTsdT
2934
40743.2
AD-
GcAucAccuGuAucuAuuAdTsdT
2935
uAAuAGAuAcAGGUGAUGCdTsdT
2936
40743.1
AD-
ccuGuAucuAuuAccuGAAdTsdT
2937
UUcAGGuAAuAGAuAcAGGdTsdT
2938
47740.1
AD-
GuAucuAuuAccuGAAucAdTsdT
2939
UGAUUcAGGuAAuAGAuACdTsdT
2940
47746.1
AD-
GAAucAGAAcuGGGAcGuudTsdT
2941
AACGUCCcAGUUCUGAUUCdTsdT
2942
47752.1
AD-
cuGGGAcGuuAAGGuGcAudTsdT
2943
AUGcACCUuAACGUCCcAGdTsdT
2944
47758.1
AD-
cucuuuGAccGGuuGcucAdTsdT
2945
UGAGcAACCGGUcAAAGAGdTsdT
2946
47764.1
AD-
cuuuGAccGGuuGcucAuudTsdT
2947
AAUGAGcAACCGGUcAAAGdTsdT
2948
47724.1
AD-
GAccGGuuGcucAuuuucudTsdT
2949
AGAAAAUGAGcAACCGGUCdTsdT
2950
47730.1
AD-
GuGAAGccAGccuAuGccAdTsdT
2951
UGGcAuAGGCUGGCUUcACdTsdT
2952
47735.1
AD-
ccAGGuAcGccAucAcuGudTsdT
2953
AcAGUGAUGGCGuACCUGGdTsdT
2954
47741.1
AD-
ccAucAcuGucuGGuAuuudTsdT
2955
AAAuACcAGAcAGUGAUGGdTsdT
2956
47747.1
AD-
GcAGcAGccAAAGAcAAGudTsdT
2957
ACUUGUCUUUGGCUGCUGCdTsdT
2958
47753.1
AD-
cAGccAAAGAcAAGuAucAdTsdT
2959
UGAuACUUGUCUUUGGCUGdTsdT
2960
47759.1
AD-
cAGccAAAGAcAAGuAucAdTsdT
2961
UGAuACUUGUCUUUGGCUGdTsdT
2962
47759.2
AD-
cAAAGAcAAGuAucAGcuAdTsdT
2963
uAGCUGAuACUUGUCUUUGdTsdT
2964
47765.1
AD-
GAcAAGuAucAGcuAGcAudTsdT
2965
AUGCuAGCUGAuACUUGUCdTsdT
2966
47725.1
AD-
GuAucAGcuAGcAucAGGAdTsdT
2967
UCCUGAUGCuAGCUGAuACdTsdT
2968
47731.1
AD-
cAGcuAGcAucAGGAcAGAdTsdT
2969
UCUGUCCUGAUGCuAGCUGdTsdT
2970
47736.1
AD-
GcuAGcAucAGGAcAGAAAdTsdT
2971
UUUCUGUCCUGAUGCuAGCdTsdT
2972
47742.1
AD-
GAAAGGuGuccAAGuAccudTsdT
2973
AGGuACUUGGAcACCUUUCdTsdT
2974
47748.1
AD-
ccuAGuGGccAGucccAGAdTsdT
2975
UCUGGGACUGGCcACuAGGdTsdT
2976
47754.1
AD-
cuGucuGGucAuGAccccAdTsdT
2977
UGGGGUcAUGACcAGAcAGdTsdT
2978
47760.1
AD-
GucuGGucAuGAccccAuudTsdT
2979
AAUGGGGUcAUGACcAGACdTsdT
2980
47766.1
AD-
cuGGGAGGAGGcAuuGucAdTsdT
2981
UGAcAAUGCCUCCUCCcAGdTsdT
2982
47726.1
AD-
GGAGGAGGcAuuGucAcuudTsdT
2983
AAGUGAcAAUGCCUCCUCCdTsdT
2984
47732.1
AD-
GcAuuGucAcuucccAccAdTsdT
2985
UGGUGGGAAGUGAcAAUGCdTsdT
2986
47737.1
AD-
GGAcuuGGGGuuGAGGuGAdTsdT
2987
UcACCUcAACCCcAAGUCCdTsdT
2988
47743.1
AD-
cucuuGcuGGcAAuGGGGudTsdT
2989
ACCCcAUUGCcAGcAAGAGdTsdT
2990
47749.1
AD-
ccAGccuGGAAuGuGAAGudTsdT
2991
ACUUcAcAUUCcAGGCUGGdTsdT
2992
47755.1
AD-
GGcAGAGuAAAAGGuGccAdTsdT
2993
UGGcACCUUUuACUCUGCCdTsdT
2994
47761.1
TABLE 13C
Human EGNL3 Single Strands and Duplex Sequences
For all the sequences in the list, ‘endolight’ chemistry was
applied as described above.
SEQ
SEQ
Duplex
Sequence (5′ to 3′)
ID
Sequence (5′ to 3′)
ID
Number
Sense
NO
Antisense
NO
AD-
GuGGcAGccGcAGGuuucudTsdT
2995
AGAAACCUGCGGCUGCcACdTsdT
2996
47767.1
AD-
GcAGccGcAGGuuucuGAAdTsdT
2997
UUcAGAAACCUGCGGCUGCdTsdT
2998
47773.1
AD-
GGcuucGcGcucGuGuAGAdTsdT
2999
UCuAcACGAGCGCGAAGCCdTsdT
3000
47779.1
AD-
GcuucGcGcucGuGuAGAudTsdT
3001
AUCuAcACGAGCGCGAAGCdTsdT
3002
47785.1
AD-
cGcGcucGuGuAGAucGuudTsdT
3003
AACGAUCuAcACGAGCGCGdTsdT
3004
47791.1
AD-
GAucccGGAccucGAuucudTsdT
3005
AGAAUCGAGGUCCGGGAUCdTsdT
3006
47797.1
AD-
cAAGGAGAGGucuAAGGcAdTsdT
3007
UGCCUuAGACCUCUCCUUGdTsdT
3008
47803.1
AD-
GGcAAuGGuGGcuuGcuAudTsdT
3009
AuAGcAAGCcACcAUUGCCdTsdT
3010
47809.1
AD-
ccGGGAAAuGGAAcAGGuudTsdT
3011
AACCUGUUCcAUUUCCCGGdTsdT
3012
47768.1
AD-
ccuGcAucuAcuAucuGAAdTsdT
3013
UUcAGAuAGuAGAUGcAGGdTsdT
3014
47786.1
AD-
GAuccuGcGGAuAuuuccAdTsdT
3015
UGGAAAuAUCCGcAGGAUCdTsdT
3016
47792.1
AD-
GGGGAAAucAuucAuAGcAdTsdT
3017
UGCuAUGAAUGAUUUCCCCdTsdT
3018
47798.1
AD-
GGAAAucAuucAuAGcAGAdTsdT
3019
UCUGCuAUGAAUGAUUUCCdTsdT
3020
47804.1
AD-
GAcAGAcuccuGuucuucudTsdT
3021
AGAAGAAcAGGAGUCUGUCdTsdT
3022
47769.1
AD-
ccuGuucuucuGGucAGAudTsdT
3023
AUCUGACcAGAAGAAcAGGdTsdT
3024
47775.1
AD-
GcAAccAGAuAuGcuAuGAdTsdT
3025
UcAuAGcAuAUCUGGUUGCdTsdT
3026
47781.1
AD-
ccAGAuAuGcuAuGAcuGudTsdT
3027
AcAGUcAuAGcAuAUCUGGdTsdT
3028
47787.1
AD-
cuAuGAcuGucuGGuAcuudTsdT
3029
AAGuACcAGAcAGUcAuAGdTsdT
3030
47793.1
AD-
GAAAuucAGGAAuuuAAcudTsdT
3031
AGUuAAAUUCCUGAAUUUCdTsdT
3032
47805.1
AD-
GAAuuuAAcuAGGAAAAcudTsdT
3033
AGUUUUCCuAGUuAAAUUCdTsdT
3034
47811.1
AD-
GccuuGuucAuuuuAGuAAdTsdT
3035
UuACuAAAAUGAAcAAGGCdTsdT
3036
47770.1
AD-
GuuccuGAAuucucuuAAAdTsdT
3037
UUuAAGAGAAUUcAGGAACdTsdT
3038
47776.1
AD-
GuuccuGAAuucucuuAAAdTsdT
3039
UUuAAGAGAAUUcAGGAACdTsdT
3040
47776.2
AD-
cuGAAuucucuuAAAuucudTsdT
3041
AGAAUUuAAGAGAAUUcAGdTsdT
3042
47782.1
AD-
cAAAGAuGGccucuucAGudTsdT
3043
ACUGAAGAGGCcAUCUUUGdTsdT
3044
47788.1
AD-
cuGcuAcuucuuGcAuccudTsdT
3045
AGGAUGcAAGAAGuAGcAGdTsdT
3046
47800.1
AD-
cccuGucuuGuGuGuGGuAdTsdT
3047
uACcAcAcAcAAGAcAGGGdTsdT
3048
47806.1
AD-
cuuGuGuGuGGuAcuucAudTsdT
3049
AUGAAGuACcAcAcAcAAGdTsdT
3050
47812.1
AD-
GuGuGGuAcuucAuGuuuudTsdT
3051
AAAAcAUGAAGuACcAcACdTsdT
3052
47771.1
AD-
GuuuucuuGccAAGAcuGudTsdT
3053
AcAGUCUUGGcAAGAAAACdTsdT
3054
47777.1
AD-
cGAGGGAAuGAAccuuAcudTsdT
3055
AGuAAGGUUcAUUCCCUCGdTsdT
3056
47783.1
AD-
cuuAcuuGcAcuuuAuGuAdTsdT
3057
uAcAuAAAGUGcAAGuAAGdTsdT
3058
47789.1
AD-
cAcuuuAuGuAuAcuuccudTsdT
3059
AGGAAGuAuAcAuAAAGUGdTsdT
3060
47795.1
AD-
GuAuAcuuccuGAuuuGAAdTsdT
3061
UUcAAAUcAGGAAGuAuACdTsdT
3062
47801.1
AD-
GGAGAAuuAucAcAAccuAdTsdT
3063
uAGGUUGUGAuAAUUCUCCdTsdT
3064
47807.1
AD-
ccuAAuGAcAuuAAuAccudTsdT
3065
AGGuAUuAAUGUcAUuAGGdTsdT
3066
47813.1
AD-
cccuGGuAGuuuuGuGuuAdTsdT
3067
uAAcAcAAAACuACcAGGGdTsdT
3068
47772.1
AD-
ccuGGuAGuuuuGuGuuAAdTsdT
3069
UuAAcAcAAAACuACcAGGdTsdT
3070
47778.1
AD-
GuGGAAAGAGcuAGGucuAdTsdT
3071
uAGACCuAGCUCUUUCcACdTsdT
3072
47784.1
AD-
cuAGGucuAcuGAuAuAcAdTsdT
3073
UGuAuAUcAGuAGACCuAGdTsdT
3074
47790.1
AD-
GucuAcuGAuAuAcAAuAAdTsdT
3075
UuAUUGuAuAUcAGuAGACdTsdT
3076
47796.1
AD-
cAuGuGuGcAucuuGAAcAdTsdT
3077
UGUUcAAGAUGcAcAcAUGdTsdT
3078
47802.1
AD-
GuGuGcAucuuGAAcAAuudTsdT
3079
AAUUGUUcAAGAUGcAcACdTsdT
3080
47808.1
Example 15
Studies of siRNA in an Animal Model:Hematology Measurements
Studies of the effects of siRNA agents in combination on a mouse model were performed to evaluate therapeutic outcomes and efficacy. Endpoints included hematology measurements (including red blood cell count, Hemoglobin levels, hematocrit levels, and reticulocyte levels).
Wild type C57BL/6 mice were dosed two times, at day 0 and 6. Dosing involved tail vein administration of an equal part mixture of the three siRNAs (AD-40894, AD-40773 and AD-40758) targeting EGLN1, EGLN2, and EGLN3 respectively. The study also included control groups of PBS treated control and a control group containing the luciferase siRNA AD-1955. The results are presented in Table 14.
Hematology
Hematocrit levels of the test group were measured at day 4 and 9. As can be seen from the data in Table 14, there was an increase in hematocrit in the mice treated with an equal part mixture of siRNAs as compared to the PBS and Luciferase controls. Measurements of red blood cell count, Hemoglobin, and reticulocyte levels were also made at day 4 and 9 and an increase in Hemoglobin and reticulocyte levels was observed. These data are also presented in Table 14. In the table “Hg” stands for Hemoglobin in g/dL, “HCT” stands for Hematocrit in %, “Ret” stands for Reticulocytes in %, and “RBC” stands for Red Blood Cells (×106 cells/uL).
TABLE 14
In vivo studies in an animal model
Day 4 Bleed
Day 9 Bleed
Ret
RBC
Hg
HCT
Ret
RBC
Hg
HCT
PBS
3.4
8.7
12.6
40.6
7.4
8.3
12.2
39.3
Luciferase
3.2
8.6
12.3
39.7
7.1
7.7
11.3
36.2
EGLN 1, 2, 3
10.1
9.4
13.6
45.8
12.5
10.5
15.4
52.9
Additional Hematology Studies: Day 0 and Day 5Dosing
Studies on the effects of the siRNA agents (alone or in combination) on a mouse model were performed to evaluate the effect of the siRNA agents on EPO production and erythropoiesis. Endpoints included hematology measurements (including red blood cell count, Hemoglobin levels, hematocrit levels, and reticulocyte levels). Wild type C57BL/6 mice were dosed two times, at day 0 and 5. Dosing involved tail vein administration at 0.5 mg/kg per EGLN family member, EGLN1 (AD-40894), EGLN2 (AD-40773), and EGLN3 (AD-40758). The study also included control groups of PBS treated mice and a group containing the luciferase siRNA AD-1955.
Hematology
Hematocrit levels of the test group were measured at sacrifice on day 11. The values are shown in Table 15 along with reticulocyte levels, hemoglobin levels and red blood cell count.
TABLE 15
In vivo studies in an animal model on day 11
Red
Blood
Reticulocyte
Cell
Hemoglobin
Hematocrit
PBS
3.7
8.5
12.8
40.5
Luciferase
2.9
8.7
12.9
41.7
EGLN1
8.7
10.6
15.4
52.3
EGLN2
3.8
8.7
12.4
40.1
EGLN3
3.6
8.3
12.3
40.0
EGLN1, 2
10.6
11.5
16.5
56.2
EGLN2, 3
7.6
10.0
14.8
49.3
EGLN1, 3
4.6
8.0
12.0
39.1
EGLN 1, 2, 3
12.2
11.9
16.9
58.5
Example 16
5′RACE Assay
A 5′RACE assay was used in order to monitor the cleavage site of target mRNA. The 5′RACE analysis showed that the downregulation of EGLN mRNA in the liver was specifically due to siRNA-mediated mRNA cleavage. Table 16 lists the 5′RACE primers used in this analysis.
TABLE 16
5′RACE Primers
Sequence 5′ to 3′
SEQ ID NO.
Adaptor oligo
CGACTGGAGCACGAGGACACTGACATGG
3081
Nested
GGACACTGACATGGACTGAAGGAGTAG
3082
Adaptor oligo
EGLN1 GSP
AGAGATGAAATGAACTCAGTTAGGTGACAGGTCTG
3083
EGLN1 PCR
TTGTTTCGTGTCCAGATGGAAAAGCTACTCTCCTC
3084
Round 1
EGLN1 PCR
GGCTTGAGTTCAACCCTCACACCTTTCTCACCTG
3085
Round 2
EGLN2 GSP
TATTTCTTGGCTGGCAGAACCTCCATAC
3086
EGLN2 PCR
CAGACAGTGGCAGCCCAGTCCATACACTG
3087
Round 1
EGLN2 PCR
CAGCAGAGGTCTCTCCTTGTTGCTCCTCAGTG
3088
Round 2
EGLN3 GSP
GATGTGGAAGAACTCCAATAGCTCTGAGGTC
3089
EGLN3 PCR
CAGTGCTGAATTACCAGGAAGCTTTCTATCCTCTG
3090
Round 1
EGLN3 PCR
GCAAGAAAACATGAAGTACCACAAACAAG
3091
Round 2
Example 17
Animal Model: Anemia
We next asked if EGLN siRNA could be used to treat anemia in the setting of chronic renal failure. Toward this end mice were subjected to 5/6 nephrectomy, which is a widely used model for anemia linked to renal failure, or sham operations (FIG. 23). The mice undergoing nephrectomy developed anemia, as expected, and were then randomized to receive phosphate buffered saline (PBS), control siRNA (luciferase siRNA), siRNAs targeting EglN1, EGLN1 and EGLN2, or combinations thereof. In keeping with the data described above, inactivation of EGLN1 led to a modest increase in red blood cell production, which was markedly accentuated by coinactivation of EGLN2. Treatment with EGLN1 and EGLN2 constructs restored both hemoglobin and hematocrit levels (FIG. 23 B, C). The maximal erythropoietic response, however, was observed after treatment with siRNA targeting all 3 EGLN paralogs. EglN inactivation in this model also led to an upregulation of EPO and a decrease in hepcidin mRNA levels, consistent with earlier studies using chemical hydroxylase inhibitors (FIG. 24).
Chronic inflammation can lead to anemia due, at least partly, to increased levels of hepcidin and altered iron trafficking (anemia of chronic disease). Rats with experimental arthritis induced by a polymer of a streptococcal antigen (PG-APS) have been used as a model for the anemia linked to inflammation (M. A. Coccia et al., Exp Hematol 29, 1201 (October, 2001); R. B. Sartor et al., Infect Immun 57, 1177 (April, 1989); W. J. Cromartie, J. G. Craddock, J. H. Schwab, S. K. Anderle, C. H. Yang, J Exp Med 146, 1585 (Dec. 1, 1977). In the 5/6 nephrectomy model combined inactivation of EGLN1 and EGLN2 was sufficient to induce a brisk erythropoietic response (FIG. 23) and we were able to identify siRNAs that can effectively target rat EglN1 and EglN2 (FIG. 25A-C). Treatment of anemic PG-APS rats with mixtures of siRNAs targeting both EglN1 and EglN2 decreased their hepcidin levels and corrected their anemia (FIG. 25).
These studies suggest that systemically administered siRNAs targeting the EGLN family would ameliorate anemias characterized by an absolute or relative deficiency of erythropoietin, such as anemias linked to chronic kidney disease or inflammation, in man. This approach would allow the body to produce native erythropoietin, thereby obviating the need for recombinant versions of this hormone. Moreover other hepatic changes induced by EGLN inhibition, such as decreased production of hepcidin, might enhance the effectiveness of endogenous erythropoietin and thereby lower the circulating erythropoietin levels needed to promote red blood cell production. This might be desirable if some of the cardiovascular complications of chronic erythropoietin production are more tightly linked to circulating erythropoietin levels, especially when supraphysiological, than to red blood cell mass per se.
Example 18
Decrease of Hepatic EGLN Activity: Photon Emission Study
It has previously been shown that EGLN activity can be monitored non-invasively in mice that ubiquitously express a HIF1α-luciferase fusion protein that contains a region of HIF1α that is sufficient to be hydroxylated by EGLN and subsequently ubiquitinated by the pVHL ubiquitin ligase complex (M. Safran et al., Proc Nall Acad Sci USA 103, 105 (Jan. 3, 2006). As expected, administration of the EGLN siRNA mix to these mice decreased hepatic, but not renal, EGLN activity as determined by increased photon emission in the region of the liver, but not kidneys, following luciferin administration (See FIG. 26). Branched DNA analysis confirmed that EglN1, EglN2, and EglN3 mRNAs were decreased in the liver, but not the kidney, and was associated with an increase hepatic, but not renal, EPO mRNA production.
It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.
While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.
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13992334
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alynylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Nov 20th, 2012 12:00AM
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Oct 4th, 2011 12:00AM
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https://www.uspto.gov?id=US08314075-20121120
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Compositions and methods for inhibiting expression of huntingtin gene
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The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Huntingtin gene (HD gene), comprising an antisense strand having a nucleotide sequence which is less than 25 nucleotides in length and which is substantially complementary to at least a part of the HD gene. The invention also relates to a pharmaceutical composition comprising the dsRNA together with a pharmaceutically acceptable carrier; methods for treating diseases caused by the expression of the HD gene, or a mutant form thereof, using the pharmaceutical composition; and methods for inhibiting the expression of the huntingtin gene in a cell.
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8314075
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1. A pharmaceutical composition comprising a double stranded ribonucleic acid (dsRNA) comprising a sense strand and an antisense strand and a pharmaceutically acceptable carrier, wherein the antisense strand comprises a nucleotide sequence consisting of SEQ ID NO:793 and a 2′-O-methyl modified nucleotide at location 4 and the sense strand comprises a nucleotide sequence consisting of SEQ ID NO:792 and a 2′-O-methyl modified nucleotide at locations 2, 3, 5, 12, 13, 15, and 17.
2. The dsRNA of claim 1, wherein said dsRNA comprises at least one additional modified nucleotide.
3. The dsRNA of claim 2, wherein said modified nucleotide is chosen from the group of: a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′ phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
4. The dsRNA of claim 2, wherein said modified nucleotide is chosen from the group of: a 5-bromo-2′-deoxyuridine, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.
5. The pharmaceutical composition of claim 1, wherein said dsRNA comprises a cholesterol moiety.
6. A method for inhibiting expression of Huntingtin (HD) gene in a cell, the method comprising: (a) introducing into the cell the pharmaceutical composition of claim 2, 3, or 4; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HD gene, thereby inhibiting expression of the HD gene in the cell.
7. A method of treating or managing Huntingtin disease comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the pharmaceutical composition of claim 2, 3, or 4.
8. The method of claim 7, wherein the pharmaceutical composition is administered to the brain of the patient.
9. The method of claim 7, wherein the pharmaceutical composition is administered by intrastriatal infusion.
10. The method of claim 8, wherein administering the pharmaceutical composition to the brain causes a decrease in Huntingtin (HD) gene mRNA in the striatum.
11. The method of claim 8, wherein administering the pharmaceutical composition to the brain causes a decrease in Huntingtin (HD) gene mRNA in the cortex.
12. The method of claim 6, wherein the dsRNA of the pharmaceutical composition comprises a cholesterol moiety.
13. The method of claim 6, wherein the pharmaceutical composition is administered in vivo to a mammal.
14. The method of claim 12, wherein the pharmaceutical composition is administered in vivo to a mammal.
15. The method of claim 6, wherein the pharmaceutical composition is administered by intrastiatal infusion.
16. The method of claim 12, wherein the pharmaceutical composition is administered by intrastiatal infusion.
17. The method of claim 7, wherein the dsRNA of the pharmaceutical composition comprises a cholesterol moiety.
18. The method of claim 17, wherein the pharmaceutical composition is administered to the brain of the patient.
19. The method of claim 17, wherein the pharmaceutical composition is administered by intrastriatal infusion.
20. The method of claim 18, wherein administering the pharmaceutical composition to the brain causes a decrease in Huntingtin (HD) gene mRNA in the striatum.
21. The method of claim 18, wherein administering the pharmaceutical composition to the brain causes a decrease in Huntingtin (HD) gene mRNA in the cortex.
22. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier consists of an aqueous buffer.
23. The method of claim 6, wherein the pharmaceutically acceptable carrier consists of an aqueous buffer.
24. The method of claim 7, wherein the pharmaceutically acceptable carrier consists of an aqueous buffer.
25. The pharmaceutical composition of claim 1, wherein the antisense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21 and the sense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21.
26. The method of claim 6, wherein the antisense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21 and the sense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21.
27. The method of claim 7, wherein the antisense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21 and the sense strand comprises a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21.
28. A method of inhibiting expression of Huntingtin (HD) gene in a cell in a patient comprising administering to the brain of the patient by intrastriatal infusion a therapeutically effective amount of a pharmaceutical composition comprising a dsRNA and an aqueous buffer, the dsRNA consisting of a sense strand and antisense strand, wherein the antisense strand comprises a nucleotide sequence consisting of SEQ ID NO:793 and a 2′-O-methyl modified nucleotide at location 4 and a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21 and the sense strand comprises a nucleotide sequence consisting of SEQ ID NO:792 and said sense strand comprises a 2′-O-methyl modified nucleotide at locations 2, 3, 5, 12, 13, 15, and 17 and a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21.
29. A method of treating or managing Huntintin disease in a patient comprising administering to the brain of the patient by intrastriatal infusion a therapeutically effective amount of a pharmaceutical composition comprising a dsRNA and an aqueous buffer, the dsRNA consisting of a sense strand and antisense strand, wherein the antisense strand comprises a nucleotide sequence consisting of SEQ ID NO:793 and a 2′-O-methyl modified nucleotide at location 4 and a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21 and the sense strand comprises a nucleotide sequence consisting of SEQ ID NO:792 and a 2′-O-methyl modified nucleotide at locations 2, 3, 5, 12, 13, 15, and 17 and a phosphorothioate at the internucleotide linkage between nucleotides 20 and 21.
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RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/417,502 filed on Apr. 2, 2009, now U.S. Pat. No. 8,080,532, issued on Dec. 20, 2011, which is a continuation of U.S. patent application Ser. No. 11/944,961 filed Nov. 26, 2007, now U.S. Pat. No. 7,749,978, issued on Jul. 6, 2010, which is a continuation of U.S. patent application Ser. No. 11/588,674, filed Oct. 27, 2006, now U.S. Pat. No. 7,320,965, issued on Jan. 22, 2008 which all claim the benefit of U.S. Provisional Application No. 60/731,555, filed Oct. 28, 2005, U.S. Provisional Application No. 60/819,038, filed Jul. 7, 2006, and U.S. Provisional Application No. 60/836,040, filed Aug. 7, 2006. The contents of each of these priority applications are incorporated herein by reference in their entirety.
SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 4, 2011, is named 19545US_CRF_sequencelisting.txt, and is 303,121 bytes in size.
FIELD OF THE INVENTION
This invention relates to double-stranded ribonucleic acid (dsRNA), and its use in mediating RNA interference to inhibit the expression of the Huntingtin gene.
BACKGROUND OF THE INVENTION
Recently, double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et al.) discloses the use of a dsRNA of at least 25 nucleotides in length to inhibit the expression of genes in C. elegans. dsRNA has also been shown to degrade target RNA in other organisms, including plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.). This natural mechanism has now become the focus for the development of a new class of pharmaceutical agents for treating disorders that are caused by the aberrant regulation of genes or the expression of a mutant form of a gene.
Huntington's disease is a progressive neurodegenerative disorder characterized by motor disturbance, cognitive loss and psychiatric manifestations (Martin and Gusella, N. Engl. J. Med. 315:1267-1276 (1986). It is inherited in an autosomal dominant fashion, and affects about 1/10,000 individuals in most populations of European origin (Harper, P. S. et al., in Huntington's disease, W. B. Saunders, Philadelphia, 1991). The hallmark of Huntington's disease is a distinctive choreic movement disorder that typically has a subtle, insidious onset in the fourth to fifth decade of life and gradually worsens over a course of 10 to 20 years until death. Occasionally, Huntington's disease is expressed in juveniles typically manifesting with more severe symptoms including rigidity and a more rapid course. Juvenile onset of Huntington's disease is associated with a preponderance of paternal transmission of the disease allele. The neuropathology of Huntington's disease also displays a distinctive pattern, with selective loss of neurons that is most severe in the caudate and putamen regions of the brain. The biochemical basis for neuronal death in Huntington's disease has not yet been explained, and there is consequently no treatment effective in delaying or preventing the onset and progression of this devastating disorder.
Although an actual mechanism for Huntington's disease remains elusive, Huntington's disease has been shown to be an autosomal dominant neurodegenerative disorder caused by an expanding glutamine repeat in a gene termed IT15 or Huntingtin (HD). Although this gene is widely expressed and is required for normal development, the pathology of Huntington's disease is restricted to the brain, for reasons that remain poorly understood. The Huntingtin gene product is expressed at similar levels in patients and controls, and the genetics of the disorder suggest that the expansion of the polyglutamine repeat induces a toxic gain of function, perhaps through interactions with other cellular proteins.
Treatment for Huntington's disease is currently not available. The choreic movements and agitated behaviors may be suppressed, usually only partially, by antipsychotics (e.g., chlorpromazine 100 to 900 mg/day po or haloperidol 10 to 90 mg/day po) or reserpine begun with 0.1 mg/day po and increased until adverse effects of lethargy, hypotension, or parkinsonism occur.
Despite significant advances in the field of RNAi and Huntington's disease treatment, there remains a need for an agent that can selectively and efficiently silence the HD gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target Huntingtin gene.
SUMMARY OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the HD gene in a cell or mammal using such dsRNA. The invention also provides compositions and methods for treating diseases caused by the expression of a mutant form of the HD gene. The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length and is substantially complementary to at least part of an mRNA transcript of the HD gene.
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the HD gene. The dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a nucleotide sequence which is substantially complementary to at least part of an mRNA encoding the huntingtin protein, and the region of complementarity is less than 30 nucleotides in length. The dsRNA, upon contacting with a cell expressing the HD gene, inhibits the expression of the HD gene by at least 20%.
For example, the dsRNA molecules of the invention can be comprised of a first sequence of the dsRNA that is selected from the group consisting of the sense sequences of Tables 1, 2, 7, 8 or 10 and the second sequence is selected from the group consisting of the antisense sequences of Tables 1, 2, 7, 8 or 10. The dsRNA molecules of the invention can be comprised of naturally occurring nucleotides or can be comprised of at least one modified nucleotide, such as a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group. Alternatively, the modified nucleotide may be chosen from the group of: a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide. Preferably, the first sequence of said dsRNA is selected from the group consisting of the sense sequences of Table 2 and the second sequence is selected from the group consisting of the antisense sequences of Table 2.
In another embodiment, the invention provides a cell comprising one of the dsRNAs of the invention. The cell is preferably a mammalian cell, such as a human cell.
In another embodiment, the invention provides a pharmaceutical composition for inhibiting the expression of the HD gene in an organism, comprising one or more of the dsRNA of the invention and a pharmaceutically acceptable carrier.
In another embodiment, the invention provides a method for inhibiting the expression of the HD gene in a cell, comprising the following steps:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA comprises at least two sequences that are complementary to each other. The dsRNA comprises a sense strand comprising a first sequence and an antisense strand comprising a second sequence. The antisense strand comprises a region of complementarity which is substantially complementary to at least a part of a mRNA encoding the HD gene, and wherein the region of complementarity is less than 30 nucleotides in length and wherein the dsRNA, upon contact with a cell expressing the HD gene, inhibits expression of the HD gene by at least 20%; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HD gene, thereby inhibiting expression of the HD gene in the cell.
In another embodiment, the invention provides methods for treating, preventing or managing Huntington's disease comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of one or more of the dsRNAs of the invention.
In another embodiment, the invention provides vectors for inhibiting the expression of the HD gene in a cell, comprising a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
In another embodiment, the invention provides cell comprising a vector for inhibiting the expression of the HD gene in a cell. The vector comprises a regulatory sequence operably linked to a nucleotide sequence that encodes at least one strand of one of the dsRNA of the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. In vitro activity of the dsRNAs provided in Table 2 against endogenous human HD mRNA expression in HeLa cells.
FIG. 2. Activity of selected dsRNAs in reducing endogenous human HD protein formation in HeLa cells.
FIG. 3. Stability of selected dsRNAs in cerebrospinal fluid (CSF) at 37° C.
FIG. 4. Long-term stability of dsRNAs AL-DP-5997, AL-DP-6000, AL-DP-6001 and AL-DP-7100 in rat CSF
DETAILED DESCRIPTION OF THE INVENTION
The invention provides double-stranded ribonucleic acid (dsRNA), as well as compositions and methods for inhibiting the expression of the HD gene in a cell or mammal using the dsRNA. The invention also provides compositions and methods for treating diseases in a mammal caused by the expression of the HD gene, or a mutant form thereof, using dsRNA. dsRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.
The dsRNA of the invention comprises an RNA strand (the antisense strand) having a region which is less than 30 nucleotides in length and is substantially complementary to at least part of an mRNA transcript of the HD gene. The use of these dsRNAs enables the targeted degradation of mRNAs of genes that are implicated in Huntington Disease. Using cell-based and animal assays, the present inventors have demonstrated that very low dosages of these dsRNA can specifically and efficiently mediate RNAi, resulting in significant inhibition of expression of the HD gene. Thus, the methods and compositions of the invention comprising these dsRNAs are useful for treating Huntington disease.
The following detailed description discloses how to make and use the dsRNA and compositions containing dsRNA to inhibit the expression of a target HD gene, as well as compositions and methods for treating diseases and disorders caused by the expression of these genes. The pharmaceutical compositions of the invention comprise a dsRNA having an antisense strand comprising a region of complementarity which is less than 30 nucleotides in length and is substantially complementary to at least part of an RNA transcript of the HD gene, together with a pharmaceutically acceptable carrier (Human HD mRNA (NM-002111), mouse HD mRNA (NM—010414) and rat HD mRNA (U18650)).
Accordingly, certain aspects of the invention provide pharmaceutical compositions comprising the dsRNA of the invention together with a pharmaceutically acceptable carrier, methods of using the compositions to inhibit expression of the HD gene, and methods of using the pharmaceutical compositions to treat diseases caused by expression of a mutant form of the HD gene.
I. DEFINITIONS
For convenience, the meaning of certain terms and phrases used in the specification, examples, and appended claims, are provided below. If there is an apparent discrepancy between the usage of a term in other parts of this specification and its definition provided in this section, the definition in this section shall prevail.
“G,” “C,” “A” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base may base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine may be replaced in the nucleotide sequences of the invention by a nucleotide containing, for example, inosine. Sequences comprising such replacement moieties are embodiments of the invention.
The gene involved in Huntington's disease (IT-15) is located at the end of the short arm of chromosome 4. A mutation occurs in the coding region of this gene and produces an unstable expanded trinucleotide repeat (cytosine-adenosine-guanosine), resulting in a protein with an expanded glutamate sequence. The normal and abnormal functions of this protein (termed huntingtin) are unknown. The abnormal huntingtin protein appears to accumulate in neuronal nuclei of transgenic mice, but the causal relationship of this accumulation to neuronal death is uncertain.
By “Huntingtin” or “HD” as used herein is meant, any Huntingtin protein, peptide, or polypeptide associated with the development or maintenance of Huntington disease. The terms “Huntingtin” and “HD” also refer to nucleic acid sequences encoding any huntingtin protein, peptide, or polypeptide, such as Huntingtin RNA or Huntingtin DNA (see for example Van Dellen et al., Jan. 24, 2004, Neurogenetics). For the Examples, the HD mRNA sequences used were Human HD mRNA (NM-002111), mouse HD mRNA (NM—010414) and rat HD mRNA (U18650).
As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of the HD gene, including mRNA that is a product of RNA processing of a primary transcription product.
As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.
As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Other conditions, such as physiologically relevant conditions as may be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they may form one or more, but preferably not more than 4, 3 or 2 mismatched base pairs upon hybridization, while retaining the ability to hybridize under the conditions most relevant to their ultimate application. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, may yet be referred to as “fully complementary” for the purposes of the invention.
“Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.
The terms “complementary”, “fully complementary” and “substantially complementary” herein may be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, as will be understood from the context of their use.
As used herein, a polynucleotide which is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide which is substantially complementary to a contiguous portion of the mRNA of interest (e.g., encoding HD). For example, a polynucleotide is complementary to at least a part of a HD mRNA if the sequence is substantially complementary to a non-interrupted portion of a mRNA encoding HD.
The term “double-stranded RNA” or “dsRNA”, as used herein, refers to a ribonucleic acid molecule, or complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary, as defined above, nucleic acid strands. The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop”. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker”. The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA. In addition to the duplex structure, a dsRNA may comprise one or more nucleotide overhangs.
As used herein, a “nucleotide overhang” refers to the unpaired nucleotide or nucleotides that protrude from the duplex structure of a dsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-end of the other strand, or vice versa. “Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the dsRNA, i.e., no nucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is double-stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule.
The term “antisense strand” refers to the strand of a dsRNA which includes a region that is substantially complementary to a target sequence. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated in the terminal regions and, if present, are preferably in a terminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.
The term “sense strand,” as used herein, refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
“Introducing into a cell”, when referring to a dsRNA, means facilitating uptake or absorption into the cell, as is understood by those skilled in the art. Absorption or uptake of dsRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. The meaning of this term is not limited to cells in vitro; a dsRNA may also be “introduced into a cell”, wherein the cell is part of a living organism. In such instance, introduction into the cell will include the delivery to the organism. For example, for in vivo delivery, dsRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection.
The terms “silence” and “inhibit the expression of”, in as far as they refer to the HD gene, herein refer to the at least partial suppression of the expression of the HD gene, as manifested by a reduction of the amount of mRNA transcribed from the HD gene which may be isolated from a first cell or group of cells in which the HD gene is transcribed and which has or have been treated such that the expression of the HD gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition is usually expressed in terms of
(
mRNA
in
control
cells
)
-
(
mRNA
in
treated
cells
)
(
mRNA
in
control
cells
)
·
100
%
Alternatively, the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to HD gene transcription, e.g. the amount of protein encoded by the HD gene which is secreted by a cell, or the number of cells displaying a certain phenotype, e.g. apoptosis. In principle, HD gene silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay. However, when a reference is needed in order to determine whether a given siRNA inhibits the expression of the HD gene by a certain degree and therefore is encompassed by the instant invention, the assay provided in the Examples below shall serve as such reference.
For example, in certain instances, expression of the HD gene is suppressed by at least about 20%, 25%, 35%, or 50% by administration of the double-stranded oligonucleotide of the invention. In a preferred embodiment, the HD gene is suppressed by at least about 60%, 70%, or 80% by administration of the double-stranded oligonucleotide of the invention. In a more preferred embodiment, the HD gene is suppressed by at least about 85%, 90%, or 95% by administration of the double-stranded oligonucleotide of the invention. In a most preferred embodiment, the HD gene is suppressed by at least about 98%, 99% or more by administration of the double-stranded oligonucleotide of the invention.
As used herein, the term “treatment” refers to the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disorder, e.g., a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease. A “patient” may be a human, but can also be a non-human animal. Treatment can refer to the reduction of any one of the overt symptoms of Huntington's disease, such as dementia or psychiatric disturbances, ranging from apathy and irritability to full-blown bipolar or schizophreniform disorder, motor manifestations include flicking movements of the extremities, a lilting gait, motor impersistence (inability to sustain a motor act, such as tongue protrusion), facial grimacing, ataxia, and dystonia.
As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of Huntington's disease or an overt symptom of the disease. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of Huntington's disease, the patient's history and age, the stage of Huntington's disease, and the administration of other anti-Huntington's disease agents.
As used herein, a “pharmaceutical composition” comprises a pharmacologically effective amount of a dsRNA and a pharmaceutically acceptable carrier. As used herein, “pharmacologically effective amount,” “therapeutically effective amount” or simply “effective amount” refers to that amount of an RNA effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 25% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 25% reduction in that parameter.
The term “pharmaceutically acceptable carrier” refers to a carrier for administration of a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture medium. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives. Suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents. Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
As used herein, a “transformed cell” is a cell into which a vector has been introduced from which a dsRNA molecule may be expressed.
II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)
In one embodiment, the invention provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the HD gene in a cell or mammal, wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the HD gene, and wherein the region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said HD gene, inhibits the expression of said HD gene by at least 20%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and preferably fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the HD gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Preferably, the duplex structure is between 15 and 30, more preferably between 18 and 25, yet more preferably between 19 and 24, and most preferably between 21 and 23 base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30, more preferably between 18 and 25, yet more preferably between 19 and 24, and most preferably between 21 and 23 nucleotides in length. The dsRNA of the invention may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the HD gene is the human HD gene. In specific embodiments, the antisense strand of the dsRNA comprises the antisense sequences of Tables 1, 2, 7, 8 or 10 and the second sequence is selected from the group consisting of the sense sequences of Tables 1, 2, 7, 8 or 10.
In further embodiments, the dsRNA comprises at least one nucleotide sequence selected from the groups of sequences provided in Tables 1, 2, 7, 8 or 10. In other embodiments, the dsRNA comprises at least two sequences selected from this group, wherein one of the at least two sequences is complementary to another of the at least two sequences, and one of the at least two sequences is substantially complementary to a sequence of an mRNA generated in the expression of the HD gene. Preferably, the dsRNA comprises two oligonucleotides, wherein one oligonucleotide is described by Tables 1, 2, 7, 8 or 10 and the second oligonucleotide is described Tables 1, 2, 7, 8 or 10.
The skilled person is well aware that dsRNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer dsRNAs can be effective as well. In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 1, 2, 7, 8 or 10, the dsRNAs of the invention can comprise at least one strand of a length of minimally 21 nt. It can be reasonably expected that shorter dsRNAs comprising one of the sequences of Tables 1, 2, 7, 8 or 10 minus only a few nucleotides on one or both ends may be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs comprising a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the sequences of Tables 1, 2, 7, 8 or 10, and differing in their ability to inhibit the expression of the HD gene in a FACS assay as described herein below by not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated by the invention.
The dsRNA of the invention can contain one or more mismatches to the target sequence. In a preferred embodiment, the dsRNA of the invention contains no more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is preferable that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the HD gene, the dsRNA preferably does not contain any mismatch within the central 13 nucleotides. The methods described within the invention can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the HD gene. Consideration of the efficacy of dsRNAs with mismatches in inhibiting expression of the HD gene is important, especially if the particular region of complementarity in the HD gene is known to have polymorphic sequence variation within the population.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2 nucleotides. dsRNAs having at least one nucleotide overhang have unexpectedly superior inhibitory properties than their blunt-ended counterparts. Moreover, the present inventors have discovered that the presence of only one nucleotide overhang strengthens the interference activity of the dsRNA, without affecting its overall stability. dsRNA having only one overhang has proven particularly stable and effective in vivo, as well as in a variety of cells, cell culture mediums, blood, and serum. Preferably, the single-stranded overhang is located at the 3′-terminal end of the antisense strand or, alternatively, at the 3′-terminal end of the sense strand. The dsRNA may also have a blunt end, preferably located at the 5′-end of the antisense strand. Such dsRNAs have improved stability and inhibitory activity, thus allowing administration at low dosages, i.e., less than 5 mg/kg body weight of the recipient per day. Preferably, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and preferably two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues. Preferably, the chemical groups that can be used to modify the dsRNA include, without limitation, methylene blue; bifunctional groups, preferably bis-(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxylbenzoyl)cystamine; 4-thiouracil; and psoralen. In one preferred embodiment, the linker is a hexa-ethylene glycol linker. In this case, the dsRNA are produced by solid phase synthesis and the hexa-ethylene glycol linker is incorporated according to standard methods (e.g., Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670). In a particular embodiment, the 5′-end of the antisense strand and the 3′-end of the sense strand are chemically linked via a hexaethylene glycol linker. In another embodiment, at least one nucleotide of the dsRNA comprises a phosphorothioate or phosphorodithioate groups. The chemical bond at the ends of the dsRNA is preferably formed by triple-helix bonds. Table 2 provides examples of modified RNAi agents of the invention.
In certain embodiments, a chemical bond may be formed by means of one or several bonding groups, wherein such bonding groups are preferably poly-(oxyphosphinicooxy-1,3-propandiol)- and/or polyethylene glycol chains. In other embodiments, a chemical bond may also be formed by means of purine analogs introduced into the double-stranded structure instead of purines. In further embodiments, a chemical bond may be formed by azabenzene units introduced into the double-stranded structure. In still further embodiments, a chemical bond may be formed by branched nucleotide analogs instead of nucleotides introduced into the double-stranded structure. In certain embodiments, a chemical bond may be induced by ultraviolet light.
In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the activation of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2′-amino modifications, 2′-amino sugar modifications, 2′-F sugar modifications, 2′-F modifications, 2′-alkyl sugar modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group, preferably by a 2′-amino or a 2′-methyl group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in (Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630 and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
Conjugating a ligand to a dsRNA can enhance its cellular absorption. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane. Alternatively, the ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides. For example, cholesterol has been conjugated to various antisense oligonucleotides resulting in compounds that are substantially more active compared to their non-conjugated analogs. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103. Other lipophilic compounds that have been conjugated to oligonucleotides include 1-pyrene butyric acid, 1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand for receptor-mediated endocytosis is folic acid. Folic acid enters the cell by folate-receptor-mediated endocytosis. dsRNA compounds bearing folic acid would be efficiently transported into the cell via the folate-receptor-mediated endocytosis. Li and coworkers report that attachment of folic acid to the 3′-terminus of an oligonucleotide resulted in an 8-fold increase in cellular uptake of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15, 1540. Other ligands that have been conjugated to oligonucleotides include polyethylene glycols, carbohydrate clusters, cross-linking agents, porphyrin conjugates, and delivery peptides.
In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, antisense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
The ligand-conjugated dsRNA of the invention may be synthesized by the use of a dsRNA that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the dsRNA. This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto. The methods of the invention facilitate the synthesis of ligand-conjugated dsRNA by the use of, in some preferred embodiments, nucleoside monomers that have been appropriately conjugated with ligands and that may further be attached to a solid-support material. Such ligand-nucleoside conjugates, optionally attached to a solid-support material, are prepared according to some preferred embodiments of the methods of the invention via reaction of a selected serum-binding ligand with a linking moiety located on the 5′ position of a nucleoside or oligonucleotide. In certain instances, an dsRNA bearing an aralkyl ligand attached to the 3′-terminus of the dsRNA is prepared by first covalently attaching a monomer building block to a controlled-pore-glass support via a long-chain aminoalkyl group. Then, nucleotides are bonded via standard solid-phase synthesis techniques to the monomer building-block bound to the solid support. The monomer building block may be a nucleoside or other organic compound that is compatible with solid-phase synthesis.
The dsRNA used in the conjugates of the invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.
Teachings regarding the synthesis of particular modified oligonucleotides may be found in the following U.S. patents: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for the preparation of oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to oligonucleotides having modified backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified oligonucleotides and the preparation thereof through reductive coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases based on the 3-deazapurine ring system and methods of synthesis thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing oligonucleotides having chiral phosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides having β-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides having alkylthio groups, wherein such groups may be used as linkers to other moieties attached at any of a variety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to oligonucleotides having phosphorothioate linkages of high chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for the preparation of 2′-O-alkyl guanosine and related compounds, including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods of synthesizing 2′-fluoro-oligonucleotides.
In the ligand-conjugated dsRNA and ligand-molecule bearing sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.
When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. Oligonucleotide conjugates bearing a variety of molecules such as steroids, vitamins, lipids and reporter molecules, has previously been described (see Manoharan et al., PCT Application WO 93/07883). In a preferred embodiment, the oligonucleotides or linked nucleosides of the invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.
The incorporation of a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group in nucleosides of an oligonucleotide confers enhanced hybridization properties to the oligonucleotide. Further, oligonucleotides containing phosphorothioate backbones have enhanced nuclease stability. Thus, functionalized, linked nucleosides of the invention can be augmented to include either or both a phosphorothioate backbone or a 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-aminoalkyl, 2′-O-allyl or 2′-fluoro group,
In some preferred embodiments, functionalized nucleoside sequences of the invention possessing an amino group at the 5′-terminus are prepared using a DNA synthesizer, and then reacted with an active ester derivative of a selected ligand. Active ester derivatives are well known to those skilled in the art. Representative active esters include N-hydrosuccinimide esters, tetrafluorophenolic esters, pentafluorophenolic esters and pentachlorophenolic esters. The reaction of the amino group and the active ester produces an oligonucleotide in which the selected ligand is attached to the 5′-position through a linking group. The amino group at the 5′-terminus can be prepared utilizing a 5′-Amino-Modifier C6 reagent. In a preferred embodiment, ligand molecules may be conjugated to oligonucleotides at the 5′-position by the use of a ligand-nucleoside phosphoramidite wherein the ligand is linked to the 5′-hydroxy group directly or indirectly via a linker. Such ligand-nucleoside phosphoramidites are typically used at the end of an automated synthesis procedure to provide a ligand-conjugated oligonucleotide bearing the ligand at the 5′-terminus.
In one preferred embodiment of the methods of the invention, the preparation of ligand conjugated oligonucleotides commences with the selection of appropriate precursor molecules upon which to construct the ligand molecule. Typically, the precursor is an appropriately-protected derivative of the commonly-used nucleosides. For example, the synthetic precursors for the synthesis of the ligand-conjugated oligonucleotides of the invention include, but are not limited to, 2′-aminoalkoxy-5′-ODMT-nucleosides, 2′-6-aminoalkylamino-5′-ODMT-nucleosides, 5′-6-aminoalkoxy-2′-deoxy-nucleosides, 5′-6-aminoalkoxy-2-protected-nucleosides, 3′-6-aminoalkoxy-5′-ODMT-nucleosides, and 3′-aminoalkylamino-5′-ODMT-nucleosides that may be protected in the nucleobase portion of the molecule. Methods for the synthesis of such amino-linked protected nucleoside precursors are known to those of ordinary skill in the art.
In many cases, protecting groups are used during the preparation of the compounds of the invention. As used herein, the term “protected” means that the indicated moiety has a protecting group appended thereon. In some preferred embodiments of the invention, compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
Representative hydroxyl protecting groups, for example, are disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-2311). Further hydroxyl protecting groups, as well as other representative protecting groups, are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York, 1991, and Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
Examples of hydroxyl protecting groups include, but are not limited to, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,p′-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.
Amino-protecting groups stable to acid treatment are selectively removed with base treatment, and are used to make reactive amino groups selectively available for substitution. Examples of such groups are the Fmoc (E. Atherton and R. C. Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds., Academic Press, Orlando, 1987, volume 9, p. 1) and various substituted sulfonylethyl carbamates exemplified by the Nsc group (Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart and Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107:621).
Additional amino-protecting groups include, but are not limited to, carbamate protecting groups, such as 2-trimethylsilylethoxycarbonyl(Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups, such as phthalimido and dithiasuccinoyl. Equivalents of these amino-protecting groups are also encompassed by the compounds and methods of the invention.
Many solid supports are commercially available and one of ordinary skill in the art can readily select a solid support to be used in the solid-phase synthesis steps. In certain embodiments, a universal support is used. A universal support allows for preparation of oligonucleotides having unusual or modified nucleotides located at the 3′-terminus of the oligonucleotide. Universal Support 500 and Universal Support II are universal supports that are commercially available from Glen Research, 22825 Davis Drive, Sterling, Va. For further details about universal supports see Scott et al., Innovations and Perspectives in solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger Epton, Mayflower Worldwide, 115-124]; Azhayev, A. V. Tetrahedron 1999, 55, 787-800; and Azhayev and Antopolsky Tetrahedron 2001, 57, 4977-4986. In addition, it has been reported that the oligonucleotide can be cleaved from the universal support under milder reaction conditions when oligonucleotide is bonded to the solid support via a syn-1,2-acetoxyphosphate group which more readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan, M. J. Am. Chem. Soc. 2003, 125, 2380.
The nucleosides are linked by phosphorus-containing or non-phosphorus-containing covalent internucleoside linkages. For the purposes of identification, such conjugated nucleosides can be characterized as ligand-bearing nucleosides or ligand-nucleoside conjugates. The linked nucleosides having an aralkyl ligand conjugated to a nucleoside within their sequence will demonstrate enhanced dsRNA activity when compared to like dsRNA compounds that are not conjugated.
The aralkyl-ligand-conjugated oligonucleotides of the invention also include conjugates of oligonucleotides and linked nucleosides wherein the ligand is attached directly to the nucleoside or nucleotide without the intermediacy of a linker group. The ligand may preferably be attached, via linking groups, at a carboxyl, amino or oxo group of the ligand. Typical linking groups may be ester, amide or carbamate groups.
Specific examples of preferred modified oligonucleotides envisioned for use in the ligand-conjugated oligonucleotides of the invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined here, oligonucleotides having modified backbones or internucleoside linkages include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of the invention, modified oligonucleotides that do not have a phosphorus atom in their intersugar backbone can also be considered to be oligonucleosides.
Specific oligonucleotide chemical modifications are described below. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modifications may be incorporated in a single dsRNA compound or even in a single nucleotide thereof.
Preferred modified internucleoside linkages or backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free-acid forms are also included.
Representative United States patents relating to the preparation of the above phosphorus-atom-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; and 5,697,248, each of which is herein incorporated by reference.
Preferred modified internucleoside linkages or backbones that do not include a phosphorus atom therein (i.e., oligonucleosides) have backbones that are formed by short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatomic or heterocyclic intersugar linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
Representative United States patents relating to the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleoside units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligonucleotide, an oligonucleotide mimetic, that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.
Some preferred embodiments of the invention employ oligonucleotides with phosphorothioate linkages and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, and —O—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.
The oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Id., pages 276-278) and are presently preferred base substitutions, even more particularly when combined with 2′-methoxyethyl sugar modifications.
Representative United States patents relating to the preparation of certain of the above-noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,808,027; all of which are hereby incorporated by reference.
In certain embodiments, the oligonucleotides employed in the ligand-conjugated oligonucleotides of the invention may additionally or alternatively comprise one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl, O-, S-, or N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. a preferred modification includes 2′-methoxyethoxy [2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE] (Martin et al., Helv. Chim. Acta, 1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998, the contents of which are incorporated by reference.
Other preferred modifications include 2′-methoxy (2′-O—CH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides.
As used herein, the term “sugar substituent group” or “2′-substituent group” includes groups attached to the 2′-position of the ribofuranosyl moiety with or without an oxygen atom. Sugar substituent groups include, but are not limited to, fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula (O-alkyl)m, wherein m is 1 to about 10. Preferred among these polyethers are linear and cyclic polyethylene glycols (PEGs), and (PEG)-containing groups, such as crown ethers and those which are disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9:93); Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardo et. al. (Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9:249), each of which is hereby incorporated by reference in its entirety. Further sugar modifications are disclosed by Cook (Anti-Huntingtin disease Drug Design, 1991, 6:585-607). Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino substitution is described in U.S. Pat. No. 6,166,197, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2′ and 5′ Substitutions,” hereby incorporated by reference in its entirety.
Additional sugar substituent groups amenable to the invention include 2′-SR and 2′-NR2 groups, wherein each R is, independently, hydrogen, a protecting group or substituted or unsubstituted alkyl, alkenyl, or alkynyl. 2′-SR Nucleosides are disclosed in U.S. Pat. No. 5,670,633, hereby incorporated by reference in its entirety. The incorporation of 2′-SR monomer synthons is disclosed by Hamm et al. (J. Org. Chem., 1997, 62:3415-3420). 2′-NR nucleosides are disclosed by Goettingen, M., J. Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett., 1996, 37, 3227-3230. Further representative 2′-substituent groups amenable to the invention include those having one of formula I or II:
wherein,
E is C1-C10 alkyl, N(Q3)(Q4) or N═C (Q3)(Q4); each Q3 and Q4 is, independently, H, C1-C10 alkyl, dialkylaminoalkyl, a nitrogen protecting group, a tethered or untethered conjugate group, a linker to a solid support; or Q3 and Q4, together, form a nitrogen protecting group or a ring structure optionally including at least one additional heteroatom selected from N and O;
q1 is an integer from 1 to 10;
q2 is an integer from 1 to 10;
q3 is 0 or 1;
q4 is 0, 1 or 2;
each Z1, Z2 and Z3 is, independently, C4-C7 cycloalkyl, C5-C14 aryl or C3-C15 heterocyclyl, wherein the heteroatom in said heterocyclyl group is selected from oxygen, nitrogen and sulfur;
Z4 is OM1, SM1, or N(M1)2; each M1 is, independently, H, C1-C8 alkyl, C1-C8 haloalkyl, C(═NH)N(H)M2, C(═O)N(H)M2 or OC(═O)N(H)M2; M2 is H or C1-C8 alkyl; and
Z5 is C1-C10 alkyl, C1-C10 haloalkyl, C2-C10 alkenyl, C2-C10 alkynyl, C6-C14 aryl, N(Q3)(Q4), OQ3, halo, SQ3 or CN.
Representative 2′-O-sugar substituent groups of formula I are disclosed in U.S. Pat. No. 6,172,209, entitled “Capped 2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in its entirety. Representative cyclic 2′-O-sugar substituent groups of formula II are disclosed in U.S. Pat. No. 6,271,358, entitled “RNA Targeted 2′-Modified Oligonucleotides that are Conformationally Preorganized,” hereby incorporated by reference in its entirety.
Sugars having O-substitutions on the ribosyl ring are also amenable to the invention. Representative substitutions for ring O include, but are not limited to, S, CH2, CHF, and CF2. See, e.g., Secrist et al., Abstract 21, Program & Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides and their Biological Applications, Park City, Utah, Sep. 16-20, 1992.
Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar. Representative United States patents relating to the preparation of such modified sugars include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; and 5,859,221, all of which are hereby incorporated by reference.
Additional modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide. For example, one additional modification of the ligand-conjugated oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more additional non-ligand moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).
Representative United States patents relating to the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; and 5,688,941, each of which is herein incorporated by reference.
The invention also includes compositions employing oligonucleotides that are substantially chirally pure with regard to particular positions within the oligonucleotides. Examples of substantially chirally pure oligonucleotides include, but are not limited to, those having phosphorothioate linkages that are at least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those having substantially chirally pure (Sp or Rp) alkylphosphonate, phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).
In certain instances, the oligonucleotide may be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of oligonucleotides bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.
Alternatively, the molecule being conjugated may be converted into a building block, such as a phosphoramidite, via an alcohol group present in the molecule or by attachment of a linker bearing an alcohol group that may be phosphitylated.
Importantly, each of these approaches may be used for the synthesis of ligand conjugated oligonucleotides. Aminolinked oligonucleotides may be coupled directly with ligand via the use of coupling reagents or following activation of the ligand as an NHS or pentfluorophenolate ester. Ligand phosphoramidites may be synthesized via the attachment of an aminohexanol linker to one of the carboxyl groups followed by phosphitylation of the terminal alcohol functionality. Other linkers, such as cysteamine, may also be utilized for conjugation to a chloroacetyl linker present on a synthesized oligonucleotide.
III. PHARMACEUTICAL COMPOSITIONS COMPRISING dsRNA
In one embodiment, the invention provides pharmaceutical compositions comprising a dsRNA, as described in the preceding section, and a pharmaceutically acceptable carrier, as described below. The pharmaceutical composition comprising the dsRNA is useful for treating a disease or disorder associated with the expression or activity of the HD gene.
In another embodiment, the invention provides pharmaceutical compositions comprising at least two dsRNAs, designed to target different regions of the HD gene, and a pharmaceutically acceptable carrier. In this embodiment, the individual dsRNAs are prepared as described in the preceding section, which is incorporated by reference herein. One dsRNA can have a nucleotide sequence which is substantially complementary to at least one part of the HD gene; additional dsRNAs are prepared, each of which has a nucleotide sequence that is substantially complementary to different part of the HD gene. The multiple dsRNAs may be combined in the same pharmaceutical composition, or formulated separately. If formulated individually, the compositions containing the separate dsRNAs may comprise the same or different carriers, and may be administered using the same or different routes of administration. Moreover, the pharmaceutical compositions comprising the individual dsRNAs may be administered substantially simultaneously, sequentially, or at preset intervals throughout the day or treatment period.
The pharmaceutical compositions of the invention are administered in dosages sufficient to inhibit expression of the HD gene. The present inventors have found that, because of their improved efficiency, compositions comprising the dsRNA of the invention can be administered at surprisingly low dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of recipient per day is sufficient to inhibit or completely suppress expression of the HD gene.
In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, more preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day, even more preferably in the range of 1.0 to 50 micrograms per kilogram body weight per day, and most preferably in the range of 1.0 to 25 micrograms per kilogram body weight per day. The pharmaceutical composition may be administered once daily, or the dsRNA may be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the day. In that case, the dsRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the dsRNA over a several day period. Sustained release formulations are well known in the art. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.
The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual dsRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.
Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as Huntington's disease. Such models are used for in vivo testing of dsRNA, as well as for determining a therapeutically effective dose.
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (including intraparenchymal and intraventricular), intrathecal, epidural, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous, intrathecal or intracranial infusion or injection.
For intramuscular, intracranial, intrathecal, subcutaneous and intravenous use, the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. In a preferred embodiment, the carrier consists exclusively of an aqueous buffer. In this context, “exclusively” means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of dsRNA in the cells that express the HD gene. Such substances include, for example, micellar structures, such as liposomes or capsids, as described below. Surprisingly, the present inventors have discovered that compositions containing only naked dsRNA and a physiologically acceptable solvent are taken up by cells, where the dsRNA effectively inhibits expression of the HD gene. Although microinjection, lipofection, viruses, viroids, capsids, capsoids, or other auxiliary agents are required to introduce dsRNA into cell cultures, surprisingly these methods and agents are not necessary for uptake of dsRNA in vivo. Aqueous suspensions according to the invention may include suspending agents such as cellulose derivatives, sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such as lecithin. Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
The pharmaceutical compositions useful according to the invention also include encapsulated formulations to protect the dsRNA against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811; PCT publication WO 91/06309; and European patent publication EP-A-43075, which are incorporated by reference herein.
Using the small interfering RNA vectors previously described, the invention also provides devices, systems, and methods for delivery of small interfering RNA to target locations of the brain. The envisioned route of delivery is through the use of implanted, indwelling, intraparenchymal catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into local brain tissue. Another envisioned route of delivery is through the use of implanted, indwelling, intraventricular catheters that provide a means for injecting small volumes of fluid containing the dsRNA of the invention directly into cerebrospinal fluid. The proximal end of these catheters may be connected to an implanted, intracerebral access port surgically affixed to the patient's cranium, or to an implanted drug pump located in the patient's torso.
Alternatively, implantable delivery devices, such as an implantable pump may be employed. Examples of the delivery devices within the scope of the invention include the Model 8506 investigational device (by Medtronic, Inc. of Minneapolis, Minn.), which can be implanted subcutaneously on the cranium, and provides an access port through which therapeutic agents may be delivered to the brain. Delivery occurs through a stereotactically implanted polyurethane catheter. Two models of catheters that can function with the Model 8506 access port include the Model 8770 ventricular catheter by Medtronic, Inc., for delivery to the intracerebral ventricles, which is disclosed in U.S. Pat. No. 6,093,180, incorporated herein by reference, and the IPA1 catheter by Medtronic, Inc., for delivery to the brain tissue itself (i.e., intraparenchymal delivery), disclosed in U.S. Ser. Nos. 09/540,444 and 09/625,751, which are incorporated herein by reference. The latter catheter has multiple outlets on its distal end to deliver the therapeutic agent to multiple sites along the catheter path. In addition to the aforementioned device, the delivery of the small interfering RNA vectors in accordance with the invention can be accomplished with a wide variety of devices, including but not limited to U.S. Pat. Nos. 5,735,814, 5,814,014, and 6,042,579, all of which are incorporated herein by reference. Using the teachings of the invention and those of skill in the art will recognize that these and other devices and systems may be suitable for delivery of small interfering RNA vectors for the treatment of neurodegenerative diseases in accordance with the invention.
In one such embodiment, the method further comprises the steps of implanting a pump outside the brain, the pump coupled to a proximal end of the catheter, and operating the pump to deliver the predetermined dosage of the at least one small interfering RNA or small interfering RNA vector through the discharge portion of the catheter. A further embodiment comprises the further step of periodically refreshing a supply of the at least one small interfering RNA or small interfering RNA vector to the pump outside said brain.
Thus, the invention includes the delivery of small interfering RNA vectors using an implantable pump and catheter, like that taught in U.S. Pat. Nos. 5,735,814 and 6,042,579, and further using a sensor as part of the infusion system to regulate the amount of small interfering RNA vectors delivered to the brain, like that taught in U.S. Pat. No. 5,814,014. Other devices and systems can be used in accordance with the method of the invention, for example, the devices and systems disclosed in U.S. Ser. Nos. 09/872,698 (filed Jun. 1, 2001) and 09/864,646 (filed May 23, 2001), which are incorporated herein by reference.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit high therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies can be used in formulation a range of dosage for use in humans. The dosage of compositions of the invention lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.
In addition to their administration individually or as a plurality, as discussed above, the dsRNAs of the invention can be administered in combination with other known agents effective in treatment of diseases. In any event, the administering physician can adjust the amount and timing of dsRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.
Methods for Treating Diseases Caused by Expression of the HD Gene
In one embodiment, the invention provides a method for treating a subject having a disease or at risk of developing a disease caused by the expression of the HD gene, or a mutant form of the HD gene. In this embodiment, the dsRNA acts as a therapeutic agent for controlling the expression of the HD protein. The method comprises administering a pharmaceutical composition of the invention to the patient (e.g., human), such that expression of the HD gene is diminished at least in part. Because of their high specificity, the dsRNAs of the invention specifically target mRNAs of the HD gene.
Neurodegenerative Diseases
Huntington's disease is also known as Huntington's Chorea, Chronic Progressive Chorea, and Hereditary Chorea. Huntington's disease is an autosomal dominant genetic disorder characterized by choreiform movements and progressive intellectual deterioration, usually beginning in middle age (35 to 50 yr). The disease affects both sexes equally. The caudate nucleus atrophies, the small-cell population degenerates, and levels of the neurotransmitters gamma-aminobutyric acid (GABA) and substance P decrease. This degeneration results in characteristic “boxcar ventricles” seen on CT scans.
The gene involved in Huntington's disease (IT-15) is located at the end of the short arm of chromosome 4. A mutation occurs in the coding region of this gene and produces an unstable expanded trinucleotide repeat (cytosine-adenosine-guanosine), resulting in a protein with an expanded glutamate sequence. The normal and abnormal functions of this protein (termed huntingtin) are unknown. The abnormal huntingtin protein appears to accumulate in neuronal nuclei of transgenic mice, but the causal relationship of this accumulation to neuronal death is uncertain.
By “Huntingtin” or “HD” as used herein is meant, any Huntingtin protein, peptide, or polypeptide associated with the development or maintenance of Huntington disease. The terms “Huntingtin” and “HD” also refer to nucleic acid sequences encoding any huntingtin protein, peptide, or polypeptide, such as Huntingtin RNA or Huntingtin DNA (see for example Van Dellen et al., Jan. 24, 2004, Neurogenetics).
Symptoms and signs develop insidiously. Dementia or psychiatric disturbances, ranging from apathy and irritability to full-blown bipolar or schizophreniform disorder, may precede the movement disorder or develop during its course. Anhedonia or asocial behavior may be the first behavioral manifestation. Motor manifestations include flicking movements of the extremities, a lilting gait, motor impersistence (inability to sustain a motor act, such as tongue protrusion), facial grimacing, ataxia, and dystonia.
Treatment for Huntington's disease is currently not available. The choreic movements and agitated behaviors may be suppressed, usually only partially, by antipsychotics (e.g., chlorpromazine 100 to 900 mg/day po or haloperidol 10 to 90 mg/day po) or reserpine begun with 0.1 mg/day po and increased until adverse effects of lethargy, hypotension, or parkinsonism occur.
Another embodiment of the present invention thus provides the use of an anti-Huntingtin dsRNA administered to a human, particularly the striatum of the human brain, for the treatment of Huntington's disease
The pharmaceutical compositions encompassed by the invention may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (including intraparenchymal and intraventricular), intrathecal, epidural, intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the pharmaceutical compositions are administered by intravenous, intrathecal or intracranial infusion or injection.
Methods for Inhibiting Expression of the HD Gene
In yet another aspect, the invention provides a method for inhibiting the expression of the HD gene in a mammal. The method comprises administering a composition of the invention to the mammal such that expression of the target HD gene is silenced. Because of their high specificity, the dsRNAs of the invention specifically target RNAs (primary or processed) of target HD gene. Compositions and methods for inhibiting the expression of these HD genes using dsRNAs can be performed as described elsewhere herein.
In one embodiment, the method comprises administering a composition comprising a dsRNA, wherein the dsRNA comprises a nucleotide sequence which is complementary to at least a part of an RNA transcript of the HD gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition may be administered by any means known in the art including, but not limited to oral or parenteral routes, including intracranial (including intraparenchymal and intraventricular), intrathecal, epidural, intravenous, intramuscular, intracranial, subcutaneous, transdermal, airway (aerosol), nasal, rectal, vaginal and topical (including buccal and sublingual) administration. In preferred embodiments, the compositions are administered by intravenous, intrathecal or intracranial infusion or injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Examples
Gene Walking of the HD Gene
ClustalW multiple alignment function of BioEdit Sequence Alignment Editor (version 7.0.4.1) was used to generate a global alignment of human (NM-002111), mouse (NM—010414) and rat (U18650) mRNA sequences.
Conserved regions were identified by embedded sequence analysis function of the software. Conserved regions were defined as sequence stretches with a minimum length of 19 bases for all aligned sequences containing no internal gaps. Sequence positions of conserved regions were counted according to the human sequence.
The siRNA design web interface at Whitehead Institute for Biomedical Research (http://jura.wi.mit.edu/siRNAext/) (Yuan et al., Nucl. Acids. Res. 2004 32:W130-W134) was used to identify all potential siRNAs targeting the conserved regions as well as their respective off-target hits to sequences in the human, mouse and rat RefSeq database. siRNAs satisfying the cross-reactivity criteria selected out of the candidates pool and subjected to the software embedded off-target analysis. For this, all selected siRNAs were analyzed in 3 rounds by the NCBI blast algorithm against the NCBI human, mouse and rat RefSeq database.
Blast results were downloaded and analyzed in order to extract the identity of the best off-target hit for the antisense strand as well as the positions of occurring mismatches. All siRNA candidates were ranked according to predicted properties. For this, different criteria were applied in order to identify siRNA with the following properties: targeting human, mouse and rat sequences (cross-reactivity given), absence of stretches with more than 3 Gs in a row, absence of human, mouse or rat predicted off-target hits. The siRNAs that contained the applied criteria were selected and synthesized (Tables 1 and 2).
As has been experienced by those working in the antisense field, ribonucleic acids are often quickly degraded by a range of nucleases present in virtually all biological environments, e.g. endonucleases, exonucleases etc. This vulnerability may be circumvented by chemically modifying these oligonucleotides such that nucleases may no longer attack. Consequently, siRNAs were synthesized with 2′-O-Methyl substitutions (Table 2) and tested for in vitro inhibitory activity on endogenous HD gene expression (HD mRNA levels).
TABLE 1
Sequences and activities of dsRNAs tested for HD gene expression inhibiting activity
SEQ
SEQ
SEQ
Remaining HD
Duplex
sequence of total
ID
Sense strand
ID
Antisense strand
ID
gene mRNA
name
19mer target site
NO:
sequence (5′-3′)
NO:
sequence (5′-3′)
NO:
[% of control]
AD-10894
gaaucgagaucggauguca
1
gaaucgagaucggaugucaTT
2
ugacauccgaucucgauucTT
3
28 ± 3
AD-10895
aaauccugcuuuagucgag
4
aaauccugcuuuagucgagTT
5
cucgacuaaagcaggauuuTT
6
45 ± 4
AD-10896
agucaguccggguagaacu
7
agucaguccggguagaacuTT
8
aguucuacccggacugacuTT
9
38 ± 2
AD-10897
gguuuaugaacugacguua
10
gguuuaugaacugacguuaTT
11
uaacgucaguucauaaaccTT
12
11 ± 2
AD-10898
guuacggguuaauuacugu
13
guuacggguuaauuacuguTT
14
acaguaauuaacccguaacTT
15
28 ± 1
AD-10899
ugcuuuagucgagaaccaa
16
ugcuuuagucgagaaccaaTT
17
uugguucucgacuaaagcaTT
18
33 ± 3
AD-10900
ucuguaccguugaguccca
19
ucuguaccguugagucccaTT
20
ugggacucaacgguacagaTT
21
35 ± 3
AD-10901
aaauuguguuagacgguac
22
aaauuguguuagacgguacTT
23
guaccgucuaacacaauuuTT
24
48 ± 6
AD-10902
uggccggaaacuugcuugc
25
uggccggaaacuugcuugcTT
26
gcaagcaaguuuccggccaTT
27
46 ± 5
AD-10903
guucaguuacggguuaauu
28
guucaguuacggguuaauuTT
29
aauuaacccguaacugaacTT
30
32 ± 3
AD-10904
gcgggcucguuccaugauc
31
gcgggcucguuccaugaucTT
32
gaucauggaacgagcccgcTT
33
31 ± 1
AD-10905
gacuccgagcacuuaacgu
34
gacuccgagcacuuaacguTT
35
acgucaagugcucggagucTT
36
28 ± 3
AD-10906
cgcauggucgacauccuug
37
cgcauggucgacauccuugTT
38
caaggaugucgaccaugcgTT
39
37 ± 2
AD-10907
aagacgagauccucgcuca
40
aagacgagauccucgcucaTT
41
ugagcgaggaucucgucuuTT
42
35 ± 1
AD-10908
aagucaguccggguagaac
43
aagucaguccggguagaacTT
44
guucuacccggacugacuuTT
45
42 ± 4
AD-10909
aaggccuucauagcgaacc
46
aaggccuucauagcgaaccTT
47
gguucgcuaugaaggccuuTT
48
65 ± 4
AD-10910
aggccuucauagcgaaccu
49
aggccuucauagcgaaccuTT
50
agguucgcuaugaaggccuTT
51
23 ± 1
AD-10911
acuccgagcacuuaacgug
52
acuccgagcacuuaacgugTT
53
cacguuaagugcucggaguTT
54
42 ± 4
AD-10912
uaacggccuucauagcgaa
55
uaaaggccuucauagcgaaTT
56
uucgcuaugaaggccuuuaTT
57
20 ± 1
AD-10913
ucugaaucgagaucggaug
58
ucugaaucgagaucggaugTT
59
cauccgaucucgauucagaTT
60
46 ± 4
AD-10914
ugaaauuguguuagacggu
61
ugaaauuguguuagacgguTT
62
accgucuaacacaauuucaTT
63
35 ± 1
AD-10915
uggcucgcauggucgacau
64
uggcucgccuggucgacauTT
65
augucgaccaugcgagccaTT
66
42 ± 5
AD-10916
aaagucaguccggguagaa
67
aaagucaguccggguagaaTT
68
uucuacccggacugacuuuTT
69
42 ± 4
AD-10917
gagugcccgugucgguucu
70
gagugcccgugucgguucuTT
71
agaaccgacacgggcacucTT
72
77 ± 8
AD-10918
ggagcucgggacggauagu
73
ggagcucgggacggauaguTT
74
acuauccgucccgagcuccTT
75
94 ± 9
AD-10919
agaaaacaagccuugccgc
76
agaaaacaagccuugccgcTT
77
gcggcaaggcuuguuuucuTT
78
43 ± 4
AD-10920
auaaucacauucguuuguu
79
auaaucacauucguuuguuTT
80
aacaaacgaaugugauuauTT
81
35 ± 4
AD-10921
ucugggcaucgcuauggaa
82
ucugggcaucgcuauggaaTT
83
uuccauagcgaugcccagaTT
84
26 ± 6
AD-10922
ggccuucauagcgaaccug
85
ggccuucauagcgaaccugTT
86
cagguucgcuaugaaggccTT
87
32 ± 12
AD-10923
cuaaaugugcucuuaggcu
88
cuaaaugugcucuuaggcuTT
89
agccuaagagcacauuuagTT
90
24 ± 8
AD-10924
guuuaugaacugacguuac
91
guuuaugaacugacguuacTT
92
guaacgucaguucauaaacTT
93
18 ± 8
AD-10925
uuuaugaacugacguuaca
94
uuuaugaacugacguuacaTT
95
uguaacgucaguucauaaaTT
96
25 ± 3
AD-10926
augaacugacguuacauca
97
augaacugacguuacaucaTT
98
ugauguaacgucaguucauTT
99
20 ± 3
AD-10927
ccacaauguugugaccgga
100
ccacaauguugugaccggaTT
101
uccggucacaacauuguggTT
102
20 ± 3
AD-10928
cugguggccgaagccguag
103
cugguggccgaagccguagTT
104
cuacggcuucggccaccagTT
105
38 ± 1
AD-10929
aauuguguuagacgguacc
106
aauuguguuagacgguaccTT
107
gguaccgucuaacacaauuTT
108
39 ± 6
AD-10930
uuguguuagacgguaccga
109
uuguguuagacgguaccgaTT
110
ucgguaccgucuaacacaaTT
111
30 ± 4
AD-10931
aaaacaagccuugccgcau
112
aaaacaagccuugccgcauTT
113
augcggcaaggcuuguuuuTT
114
32 ± 3
AD-10932
aagagcuguaccguuggga
115
aagagcuguaccguugggaTT
116
ucccaacgguacagcucuuTT
117
43 ± 5
AD-10933
auaccucagguccuguuac
118
auaccucagguccuguuacTT
119
guaacaggaccugagguauTT
120
36 ± 4
AD-10934
uccugcuuuagucgagaac
121
uccugcuuuagucgagaacTT
122
guucucgacuaaagcaggaTT
123
35 ± 7
AD-10935
cauaaucacauucguuugu
124
cauaaucacauucguuuguTT
125
acaaacgaaugugauuaugTT
126
28 ± 2
AD-10936
aagcgacugucucgacaga
127
aagcgacugucucgacagaTT
128
ucugucgagacagucgcuuTT
129
29 ± 3
AD-10937
ccgagcacuuaacguggcu
130
ccgagcacuuaacguggcuTT
131
agccacguuaagugcucggTT
132
38 ± 5
AD-10938
cuggcucgcauggucgaca
133
cuggcucgcauggucgacaTT
134
ugucgaccaugcgagccagTT
135
35 ± 2
AD-10939
uugucgccggguagaaaag
136
uugucgccggguagaaaugTT
137
cauuucuacccggcgacaaTT
138
47 ± 8
AD-10940
ugcaagacucacuuagucc
139
ugcaagacucacuuaguccTT
140
ggacuaagugagucuugcaTT
141
56 ± 9
AD-10941
gaaacagugaguccggaca
142
gcaacagugaguccggacaTT
143
uguccggacucacuguuucTT
144
36 ± 4
AD-10942
aaaucccaguguuggacca
145
aaaucccaguguuggaccaTT
146
ugguccaacacugggauuuTT
147
37 ± 4
AD-10943
gcuagcuccaugcuuaagc
148
gcuagcuccaugcuuaagcTT
149
gcuuaagcauggagcuagcTT
150
47 ± 4
AD-10944
uccaugcuuaagccuaggg
151
uccaugcuuaagccuagggTT
152
cccuaggcuuaagcauggaTT
153
102 ± 12
AD-10945
ccaugcuuaagccuaggga
154
ccaugcuuaagccuagggaTT
155
ucccuaggcuuaagcauggTT
156
34 ± 5
AD-10946
ucaacagcuacacacgugu
157
ucaacagcuacacacguguTT
158
acacguguguagcuguugaTT
159
40 ± 5
AD-10947
augugugccacugcguuuu
160
augugugccacugcguuuuTT
161
aaaacgcaguggcacacauTT
162
31 ± 3
AD-10948
ugugugccacugcguuuua
163
ugugugccacugcguuuuaTT
164
uaaaacgcaguggcacacaTT
165
33 ± 1
AD-10949
ucaguccggguagaacuuc
166
ucaguccggguagaccuucTT
167
gaaguucuacccggacugaTT
168
58 ± 5
AD-10950
aguccggguagaacuucag
169
aguccggguagaacuucagTT
170
cugaaguucuacccggacuTT
171
34 ± 3
AD-10951
gauuguugcuauggagcgg
172
gauuguugcuauggagcggTT
173
ccgcuccauagcaacaaucTT
174
46 ± 7
AD-10952
acuuguuuacgaaaugucc
175
acuuguuuacgaaauguccTT
176
ggacauuucguaaacaaguTT
177
46 ± 2
AD-10953
cuuguuuacgaaaugucca
178
cuuguuuacgaaauguccaTT
179
uggacauuucguaaacaagTT
180
30 ± 1
AD-10954
gcuuccgcacaugccgcgg
181
gcuuccgcacaugccgcggTT
182
ccgcggcaugugcggaagcTT
183
45 ± 5
AD-10955
uaauuuuaacguaacucuu
184
uaauuuuaacguaacucuuTT
185
aagaguuacguuaaaauuaTT
186
104 ± 6
AD-10956
cuuucuaugcccguguaaa
187
cuuucuaugcccguguaaaTT
188
uuuacacgggcauagaaagTT
189
59 ± 3
AD-10957
aaagggaaggacugacgag
190
aaagggaaggacugacgagTT
191
cucgucaguccuucccuuuTT
192
84 ± 4
AD-10958
gcuggcucgcauggucgac
193
gcuggcucgcauggucgacTT
194
gucgaccaugcgagccagcTT
195
44 ± 4
AD-10959
ugacguuacaucauacaca
196
ugacguuacaucauacacaTT
197
uguguaugauguaacgucaTT
198
19 ± 3
AD-10960
acgguaccgacaaccagua
199
acgguaccgacaaccaguaTT
200
uacugguugucgguaccguTT
201
25 ± 3
AD-10961
gguaccgacaaccaguauu
202
gguaccgacaaccaguauuTT
203
aauacugguugucgguaccTT
204
19 ± 3
AD-10962
acgagugcucaauaauguu
205
acgagugcucaauaauguuTT
206
aacauuauugagcacucguTT
207
19 ± 3
AD-10963
caucggagaguuucugucc
208
caucggagaguuucuguccTT
209
ggacagaaacucuccgaugTT
210
38 ± 5
AD-10964
gcgaaccugaagucaagcu
211
gcgaaccugaagucaagcuTT
212
agcuugacuucagguucgcTT
213
35 ± 4
AD-10965
cugaaucgagaucggaugu
214
cugaaucgagaucggauguTT
215
acauccgaucucgauucagTT
216
31 ± 2
AD-10966
cgguaccgacaaccaguau
217
cgguaccgacaaccaguauTT
218
auacugguugucgguaccgTT
219
26 ± 2
AD-10967
acugaaccgggugaucaag
220
acugaaccgggugaucaagTT
221
cuugaucacccgguucaguTT
222
43 ± 3
AD-10968
ccuugccgcaucaaaggug
223
ccuugccgcaucaaaggugTT
224
caccuuugaugcggcaaggTT
225
64 ± 9
AD-10969
cuuuggcggauugcauucc
226
cuu+ggcggauugcauuccTT
227
ggaaugcaauccgccaaagTT
228
45 ± 3
AD-10970
cuguaccguugagucccaa
229
cuguaccguugagucccaaTT
230
uugggacucaacgguacagTT
231
33 ± 1
AD-10971
uguaccguugagucccaag
232
uguaccguugagucccaagTT
233
cuugggacucaacgguacaTT
234
36 ± 4
AD-10972
agucgagaaccaaugaugg
235
agucgagaaccaaugauggTT
236
ccaucauugguucucgacuTT
237
34 ± 5
AD-10973
ccgacuaccgcuggugggc
238
ccgacuaccgcuggugggcTT
239
gcccaccagcgguagucggTT
240
47 ± 7
AD-10974
auaucaccggcugcugacu
241
auaucaccggcugcugacuTT
242
agucagcagccggugauauTT
243
73 ± 6
AD-10975
ugcauaucgcugggcucaa
244
ugcauaucgcugggcucaaTT
245
uugagcccagcgauaugcaTT
246
88 ± 1
AD-10976
uuguuuacgacgugaucua
247
uuguuuacgacgugaucuaTT
248
uagaucacgucguaaacaaTT
249
66 ± 5
AD-10977
guguuagacgguaccgaca
250
guguuagacgguaccgacaTT
251
ugucgguaccgucuaacacTT
252
21 ± 2
AD-10978
cuugaacuacaucgaucau
253
cuugaacuacaucgaucauTT
254
augaucgauguaguucaagTT
255
37 ± 6
AD-10979
ggccggaaacuugcuugca
256
ggccggaaacuugcuugcaTT
257
ugcaagcaaguuuccggccTT
258
32 ± 3
AD-10980
cugucucgacagauagcug
259
cugucucgacagauagcugTT
260
cagcuaucugucgagacagTT
261
26 ± 8
AD-10981
gcaucgcuauggaacuuuu
262
gcaucgcuauggaacuuuuTT
263
aaaaguuccauagcgaugcTT
264
11 ± 2
AD-10982
acugacguuacaucauaca
265
acugacguuacaucauacaTT
266
uguaugauguaacgucaguTT
267
13 ± 4
AD-10983
cugacguuacaucauacac
268
cugacguuacaucauacacTT
269
guguaugauguaacgucagTT
270
31 ± 5
AD-10984
ugaaucgagaucggauguc
271
ugaaucgagaucggaugucTT
272
gacauccgaucucgauucaTT
273
62 ± 13
AD-10985
uagacgguaccgacaacca
274
ucgacgguaccgacaaccaTT
275
ugguugucgguaccgucuaTT
276
30 ± 4
AD-10986
uugccgcaucaaaggugac
277
uugccgcaucaaaggugacTT
278
gucaccuuugaugcggcaaTT
279
68 ± 6
AD-10987
aacuacaucgaucauggag
280
aacuacaucgaucauggagTT
281
cuccaugaucgauguaguuTT
282
61 ± 5
AD-10988
uuuggcggauugcauuccu
283
uuuggcggauugcauuccuTT
284
aggaaugcaauccgccaaaTT
285
48 ± 5
AD-10989
gcuuuagucgagaaccaau
286
gcuuuagucgagaaccaauTT
287
auugguucucgacuaaagcTT
288
29 ± 3
AD-10990
uuuagucgagaaccaauga
289
uuuagucgagaaccaaugaTT
290
ucauugguucucgacuaaaTT
291
29 ± 1
AD-10991
uagucgagaaccaaugaug
292
uagucgagaaccaaugaugTT
293
caucauugguucucgacuaTT
294
36 ± 3
AD-10992
aagugucuacccaguugaa
295
aagugucuacccaguugaaTT
296
uucaacuggguagacacuuTT
297
31 ± 3
AD-10993
ucaguuacggguuaauuac
298
ucaguuacggguuaauuacTT
299
guaauuaacccguaacugaTT
300
44 ± 8
AD-10994
uuacggguuaauuacuguc
301
uuacggguuaauuacugucTT
302
gacaguaauuaacccguaaTT
303
88 ± 17
AD-10995
uacggguuaauuacugucu
304
uacggguuaauuacugucuTT
305
agacaguaauuaacccguaTT
306
65 ± 5
AD-10996
gucucgacagauagcugac
307
gucucgacagauagcugacTT
308
gucagcuaucugucgagacTT
309
32 ± 3
AD-10997
ucucgacagauagcugaca
310
ucucgacagauagcugacaTT
311
ugucagcuaucugucgagaTT
312
34 ± 2
AD-10998
ugcgggcucguuccaugau
313
ugcgggcucguuccaugauTT
314
aucauggaacgagcccgcaTT
315
34 ± 4
AD-10999
uucagucucguugugaaaa
316
uucagucucguugugaaaaTT
317
uuuucacaacgagacugaaTT
318
37 ± 2
AD-11000
ugucgccggguagaaaugc
319
ugucgccggguagaaaugcTT
320
gcauuucuacccggcgacaTT
321
91 ± 2
AD-11001
ucggaguucaaccuaagcc
322
ucggaguucaaccuaagccTT
323
ggcuuagguugaacuccgaTT
324
70 ± 6
AD-11002
caugcuuaagccuagggau
325
caugcuuaagccuagggauTT
326
aucccuaggcuuaagcaugTT
327
37 ± 6
AD-11003
ccgcugagucuggaucucc
328
ccgcugagucuggaucuccTT
329
ggagauccagacucagcggTT
330
70 ± 12
AD-11004
ugucaacagcuacacacgu
331
ugucaacagcuacacacguTT
332
acguguguagcuguugacaTT
333
43 ± 4
AD-11005
guggccggcaacccagcug
334
guggccggcaacccagcugTT
335
cagcuggguugccggccacTT
336
40 ± 3
AD-11006
gaaagggaucgcccacugc
337
gaaagggaucgcccacugcTT
338
gcagugggcgaucccuuucTT
339
42 ± 2
AD-11007
aaagggaucgcccacugcg
340
aaagggaucgcccacugcgTT
341
cgcagugggcgaucccuuuTT
342
43 ± 2
AD-11008
cggguagaacuucagaccc
343
cggguagaacuucagacccTT
344
gggucugaaguucuacccgTT
345
33 ± 3
AD-11009
gcucgaccgcagggccuuc
346
gcucgaccgcagggccuucTT
347
gaaggcccugcggucgagcTT
348
49 ± 4
AD-11010
agcccauaucaccggcugc
349
agcccauaucaccggcugcTT
350
gcagccggugauaugggcuTT
351
46 ± 1
AD-11011
uucuaugcccguguaaagu
352
uucuaugcccguguaaaguTT
353
acuuuacacgggcauaaaaTT
354
100 ± 5
AD-11012
cccuuuuagucaggagagu
355
cccuuuuagucaggagaguTT
356
acucuccugacuaaaagggTT
357
94 ± 8
AD-11013
gguuggcgacugucaugug
358
gguuggcgacugucaugugTT
359
cacaugacagucgccaaccTT
360
156 ± 10
AD-11014
acugucucgacagauagcu
361
acugucucgacagauagcuTT
362
agcuaucugucgagacaguTT
363
39 ± 5
AD-11015
uugucugacaauaugugaa
364
uugucugacaauaugugaaTT
365
uucacauauugucagacaaTT
366
21 ± 1
AD-11016
cugggcaucgcuauggaac
367
cugggcaucgcuauggaacTT
368
guuccauagcgaugcccagTT
369
25 ± 3
AD-11017
cucggaguuugcgugcugc
370
cucggaguuugcgugcugcTT
371
gcagcacgcaaacuccgagTT
372
29 ± 3
AD-11018
uguuaaaggccuucauagc
373
uguuaaaggccuucauagcTT
374
gcuaugaaggccuuuaacaTT
375
42 ± 3
AD-11019
uuaaaggccuucauagcga
376
uuaaaggccuucauagcgaTT
377
ucgcuaugaaggccuuuaaTT
378
32 ± 4
AD-11020
gccuucauagcgaaccuga
379
gccuucauagcgaaccugaTT
380
ucagguucgcuaugaaggcTT
381
26 ± 10
AD-11021
aaggcagcuucggagugac
382
aaggcagcuucggagugacTT
383
gucacuccgaagcugccuuTT
384
27 ± 2
AD-11022
agguuuaugaacugacguu
385
agguuuaugaacugacguuTT
386
aacgucaguucauaaaccuTT
387
10 ± 2
AD-11023
aacugacguuacaucauac
388
aacugacguuacaucauacTT
389
guaugauguaacgucaguuTT
390
39 ± 3
AD-11024
cacaauguugugaccggag
391
cacaauguugugaccggagTT
392
cuccggucacaacauugugTT
393
23 ± 4
AD-11025
caauguugugaccggagcc
394
caauguugugaccggagccTT
395
ggcuccggucacaacauugTT
396
25 ± 4
AD-11026
agcagcucuucagaacgcc
397
agcagcucuucagaacgccTT
398
ggcguucugaagagcugcuTT
399
74 ± 11
AD-11027
guggccgaagccguagugg
400
guggccgaagccguaguggTT
401
ccacuacggcuucggccacTT
402
32 ± 4
AD-11028
cguagugggaguauugugg
403
cguagugggaguauuguggTT
404
ccacaauacucccacuacgTT
405
26 ± 4
AD-11029
ggaguauuguggaacuuau
406
ggaguauuguggaacuuauTT
407
auaaguuccacaauacuccTT
408
20 ± 2
AD-11030
aguauuguggaacuuauag
409
aguauuguggaacuuauagTT
410
cuauaaguuccacaauacuTT
411
35 ± 3
AD-11031
gagaucggaugucagcagc
412
gagaucggaugucagcagcTT
413
gcugcugacauccgaucucTT
414
53 ± 18
AD-11032
cagcgccgucccaucugac
415
cagcgccgucccaucugacTT
416
gucagaugggacggcgcugTT
417
49 ± 4
AD-11033
ccaccgaagggccugauuc
418
ccaccgaagggccugauucTT
419
gaaucaggcccuucgguggTT
420
28 ± 6
AD-11034
auuguguuagacgguaccg
421
auuguguuagacgguaccgTT
422
cgguaccgucuaacacaauTT
423
111 ± 12
AD-11035
ccgacaaccaguauuuggg
424
ccgacaaccaguauuugggTT
425
cccaaauacugguugucggTT
426
25 ± 5
AD-11036
aaacaagccuugccgcauc
427
aaacaagccuugccgcaucTT
428
gaugcggcaaggcuuguuuTT
429
35 ± 4
AD-11037
gccuugccgcaucaaaggu
430
gccuugccgcaucaaagguTT
431
accuuugaugcggcaaggcTT
432
36 ± 9
AD-11038
aucuugaacuacaucgauc
433
aucuugaacuacaucgaucTT
434
gaucgauguaguucaagauTT
435
40 ± 5
AD-11039
aucgaucauggagacccac
436
aucgaucauggagacccacTT
437
gugggucuccaugaucgauTT
438
69 ± 5
AD-11040
uggagacccacagguucga
439
uggagacccacagguucgaTT
440
ucgaaccugugggucuccaTT
441
39 ± 9
AD-11041
ggagacccacagguucgag
442
ggagacccacagguucgagTT
443
cucgaaccugugggucuccTT
444
65 ± 14
AD-11042
ccgcuuccacgugggagau
445
ccgcuuccacgugggagauTT
446
aucucccacguggaagcggTT
447
63 ± 2
AD-11043
ucuuuggcggauugcauuc
448
ucuuuggcggauugcauucTT
449
gaaugcaauccgccaaagaTT
450
60 ± 5
AD-11044
uuggcggauugcauuccuu
451
uuggcggauugcauuccuuTT
452
aaggaaugcaauccgccaaTT
453
30 ± 2
AD-11045
agcagcuacagugaguuag
454
agcagcuacagugaguuagTT
455
cuaacucacuguagcugcuTT
456
64 ± 2
AD-11046
cgagugcucaauaauguug
457
cgagugcucaauaauguugTT
458
caacauuauugagcacucgTT
459
18 ± 5
AD-11047
aauuaggcuugucccaaag
460
aauuaggcuugucccaaagTT
461
cuuugggacaagccuaauuTT
462
54 ± 14
AD-11048
uggaguuuagguuggcacu
463
uggaguuuagguuggcacuTT
464
agugccaaccuaaacuccaTT
465
44 ± 5
AD-11049
cuugguucccauuggaucu
466
cuugguucccauuggaucuTT
467
agauccaaugggaaccaagTT
468
32 ± 4
AD-11050
uuuuggccggaaacuugcu
469
uuuuggccggaaacuugcuTT
470
agcaaguuuccggccaaaaTT
471
53 ± 12
AD-11051
ugccuucucuaacaaaccc
472
ugccuucucuaacaaacccTT
473
ggguuuguuagagaaggcaTT
474
57 ± 5
AD-11052
uaagucccauccgacgaaa
475
uaagucccauccgacgaaaTT
476
uuucgucggaugggacuuaTT
477
43 ± 4
AD-11053
ugauaccucagguccuguu
478
ugauaccucagguccuguuTT
479
aacaggaccugagguaucaTT
480
26 ± 2
AD-11054
gauaccucagguccuguua
481
gauaccucagguccuguuaTT
482
uaacaggaccugagguaucTT
483
30 ± 5
AD-11055
uguuacaacaaguaaaucc
484
uguuacaacaaguacauccTT
485
ggauuuacuuguuguaacaTT
486
81 ± 4
AD-11056
cuaggauaccugaaauccu
487
cuaggauaccugaaauccuTT
488
aggauuucagguauccuagTT
489
35 ± 13
AD-11057
cuuuagucgagaaccaaug
490
cuuuagucgagaaccaaugTT
491
cauugguucucgacuaaagTT
492
33 ± 6
AD-11058
acuguuuguguucaacaau
493
acuguuuguguucaacaauTT
494
auuguugaacacaaacaguTT
495
39 ± 4
AD-11059
caauuguugaagacucucu
496
caauuguugaagacucucuTT
497
agagagucuucaacaauugTT
498
39 ± 3
AD-11060
caagucacaaggccgagca
499
caagucacaaggccgagcaTT
500
ugcucggccuugugacuugTT
501
40 ± 1
AD-11061
aagucacaaggccgagcac
502
aagucacaaggccgagcacTT
503
gugcucggccuugugacuuTT
504
38 ± 5
AD-11062
ggcuuguaccacuacugcu
505
ggcuuguaccacuacugcuTT
506
agcaguagugguacaagccTT
507
27 ± 3
AD-11063
acgacaccucgggaugguu
508
acgacaccucgggaugguuTT
509
aaccaucccgaggugucguTT
510
38 ± 4
AD-11064
caccucgggaugguuugau
511
caccucgggaugguuugauTT
512
aucaaaccaucccgaggugTT
513
52 ± 11
AD-11065
cucgggaugguuugauguc
514
cucgggaugguuugaugucTT
515
gacaucaaaccaucccgagTT
516
49 ± 13
AD-11066
agugucacaaagaaccgug
517
agugucacaaagaaccgugTT
518
cacgguucuuugugacacuTT
519
43 ± 13
AD-11067
gugucacaaagaaccgugc
520
gugucacaaagaaccgugcTT
521
gcacgguucuuugugacacTT
522
30 ± 6
AD-11068
aaccgugcagauaagaaug
523
aaccgugcagauaagaaugTT
524
cauucuuaucugcacgguuTT
525
36 ± 7
AD-11069
accgugcagauaagaaugc
526
accgugcagauaagaaugcTT
527
gcauucuuaucugcacgguTT
528
39 ± 3
AD-11070
ccgugcagauaagaaugcu
529
ccgugcagauaagaaugcuTT
530
agcauucuuaucugcacggTT
531
39 ± 3
AD-11071
gcagauaagaaugcuauuc
532
gcagauaagaaugcuauucTT
533
gaauagcauucuuaucugcTT
534
37 ± 4
AD-11072
acauucguuuguuugaacc
535
acauucguuuguuugaaccTT
536
gguucaaacaaacgaauguTT
537
62 ± 3
AD-11073
ugaaccucuuguuauaaaa
538
ugaaccucuuguuauaaaaTT
539
uuuuauaacaagagguucaTT
540
21 ± 4
AD-11074
uuuagauuugcuggcgcag
541
uuuagauuugcuggcgcagTT
542
cugcgccagcaaaucuaaaTT
543
80 ± 5
AD-11075
ugguucaguuacggguuaa
544
ugguucaguuacggguuaaTT
545
uuaacccguaacugaaccaTT
546
32 ± 13
AD-11076
gggccaguucagggaauca
547
gggccaguucagggaaucaTT
548
ugauucccugaacuggcccTT
549
30 ± 7
AD-11077
uggaagcgacugucucgac
550
uggaagcgacugucucgacTT
551
gucgagacagucgcuuccaTT
552
41 ± 5
AD-11078
ggaagcgacugucucgaca
553
ggaagcgacugucucgacaTT
554
ugucgagacagucgcuuccTT
555
30 ± 8
AD-11079
gaagcgacugucucgacag
556
gaagcgacugucucgacagTT
557
cugucgagacagucgcuucTT
558
35 ± 8
AD-11080
gcgacugucucgacagaua
559
gcgacugucucgacagauaTT
560
uaucugucgagacagucgcTT
561
35 ± 6
AD-11081
ugucucgacagauagcuga
562
ugucucgacagauagcugaTT
563
ucagcuaucugucgagacaTT
564
33 ± 4
AD-11082
cucgacagauagcugacau
565
cucgacagauagcugacauTT
566
augucagcuaucugucgagTT
567
39 ± 7
AD-11083
agguggaaaugagugagca
568
agguggaaaugagugagcaTT
569
ugcucacucauuuccaccuTT
570
27 ± 4
AD-11084
agugagcagcaacauacuu
571
agugagcagcaacauacuuTT
572
aaguauguugcugcucacuTT
573
23 ± 3
AD-11085
guuccgcagugauggcugu
574
guuccgcagugauggcuguTT
575
acagccaucacugcggaacTT
576
37 ± 4
AD-11086
caaccacaccgacuaccgc
577
caaccacaccgacuaccgcTT
578
gcgguagucggugugguugTT
579
36 ± 5
AD-11087
aaccacaccgacuaccgcu
580
anccacaccgacuaccgcuTT
581
agcgguagucggugugguuTT
582
48 ± 10
AD-11088
accacaccgacuaccgcug
583
accacaccgacuaccgcugTT
584
cagcgguagucggugugguTT
585
42 ± 3
AD-11089
cccgaaaagacacagucug
586
cccgaaaagacacagucugTT
587
cagacugugucuuuucgggTT
588
37 ± 2
AD-11090
uccagcacaaaguuacuua
589
uccagcacaaaguuacuuaTT
590
uaaguaacuuugugcuggaTT
591
35 ± 4
AD-11091
uuggaaugugcaauagaga
592
uuggaaugugcaauagagaTT
593
ucucuauugcacauuccaaTT
594
29 ± 6
AD-11092
agaucugaucagccuuucc
595
agaucugaucagccuuuccTT
596
ggaaaggcugaucagaucuTT
597
43 ± 3
AD-11093
caggcaauucagucucguu
598
caggcaauucagucucguuTT
599
aacgagacugaauugccugTT
600
31 ± 3
AD-11094
ggcaauucagucucguugu
601
ggcaauucagucucguuguTT
602
acaacgagacugaauugccTT
603
27 ± 3
AD-11095
gcaauucagucucguugug
604
gcaauucagucucguugugTT
605
cacaacgagacugaauugcTT
606
23 ± 3
AD-11096
aauucagucucguugugaa
607
aauucagucucguugugaaTT
608
uucacaacgagacugaauuTT
609
27 ± 3
AD-11097
ucagucucguugugaaaac
610
ucagucucguugugaaaacTT
611
guuuucacaacgagacugaTT
612
42 ± 8
AD-11098
aaaccuuucaacuccaacc
613
aaaccuuucaacuccaaccTT
614
gguuggaguugaaagguuuTT
615
60 ± 7
AD-11099
cuuuccgugugcuggcucg
616
cuuuccgugugcuggcucgTT
617
cgagccagcacacggaaagTT
618
46 ± 4
AD-11100
ccgugugcuggcucgcaug
619
ccgugugcuggcucgcaugTT
620
caugcgagccagcacacggTT
621
33 ± 3
AD-11101
ucgacauccuugcuugucg
622
ucgacauccuugcuugucgTT
623
cgacaagcaaggaugucgaTT
624
47 ± 4
AD-11102
ugcuugucgccggguagaa
625
ugcuugucgccggguagaaTT
626
uucuacccggcgacaagcaTT
627
43 ± 8
AD-11103
gcuugucgccggguagaaa
628
gcuugucgccggguagaaaTT
629
uuucuacccggcgacaagcTT
630
35 ± 7
AD-11104
cuugucgccggguagaaau
631
cuugucgccggguagaaauTT
632
auuucuacccggcgacaagTT
633
37 ± 9
AD-11105
ggcccaguugccaauggaa
634
ggcccaguugccaauggaaTT
635
uuccauuggcaacugggccTT
636
39 ± 5
AD-11106
cagguuucgucucuccacc
637
cagguuucgucucuccaccTT
638
gguggagagacgaaaccugTT
639
38 ± 8
AD-11107
ggcacgugucacuggaaac
640
ggcacgugucacuggaaacTT
641
guuuccagugacacgugccTT
642
39 ± 3
AD-11108
cuggaaacagugaguccgg
643
cuggaaacagugaguccggTT
644
ccggacucacuguuuccagTT
645
51 ± 3
AD-11109
caaaucccaguguuggacc
646
caaaucccaguguuggaccTT
647
gguccaacacugggauuugTT
648
53 ± 4
AD-11110
acucggaguucaaccuaag
649
acucggaguucaaccuaagTT
650
cuuagguugaacuccgaguTT
651
43 ± 3
AD-11111
cucggaguucaaccuaagc
652
cucggaguucaaccuaagcTT
653
gcuuagguugaacuccgagTT
654
41 ± 6
AD-11112
agccuagggaugagugaaa
655
agccuagggaugagugaaaTT
656
uuucacucaucccuaggcuTT
657
34 ± 5
AD-11113
gucaacagcuacacacgug
658
gucaacagcuacacacgugTT
659
cacguguguagcuguugacTT
660
42 ± 4
AD-11114
gauggucacccaaaccggg
661
gauggucacccaaaccgggTT
662
cccgguuugggugaccaucTT
663
49 ± 3
AD-11115
ugacagaacugcgaagggu
664
ugacagnacugcgaaggguTT
665
acccuucgcaguucugucaTT
666
53 ± 8
AD-11116
gaagacgagauccucgcuc
667
gaagacgagauccucgcucTT
668
gagcgaggaucucgucuucTT
669
43 ± 7
AD-11117
acgagauccucgcucagua
670
acgagauccucgcucaguaTT
671
uacugagcgaggaucucguTT
672
40 ± 9
AD-11118
aaccugaaagggaucgccc
673
aaccugaaagggaucgcccTT
674
gggcgaucccuuucagguuTT
675
81 ± 7
AD-11119
gaucgcccacugcgugaac
676
gaucgcccacugcgugaacTT
677
guucacgcagugggcgaucTT
678
50 ± 7
AD-11120
cacugcgugaacauucaca
679
cacugcgugaacauucacaTT
680
ugugaauguucacgcagugTT
681
40 ± 13
AD-11121
agaacuauccucuggacgu
682
agaacuauccucuggacguTT
683
acguccagaggauaguucuTT
684
41 ± 8
AD-11122
gucaguccggguagaacuu
685
gucaguccggguagaacuuTT
686
aaguucuacccggacugacTT
687
37 ± 10
AD-11123
ugaacaaagucaucggaga
688
ugaacaaagucaucggagaTT
689
ucuccgaugacuuuguucaTT
690
39 ± 6
AD-11124
aagucaucggagaguuucu
691
aagucaucggagaguuucuTT
692
agaaacucuccgaugacuuTT
693
40 ± 2
AD-11125
gucaucggagaguuucugu
694
gucaucggagaguuucuguTT
695
acagaaacucuccgaugacTT
696
37 ± 4
AD-11126
ggccaccgugguguauaag
697
ggccaccgugguguauaagTT
698
cuuauacaccacgguggccTT
699
48 ± 2
AD-11127
accgugguguauaaggugu
700
accgugguguauaagguguTT
701
acaccuuauacaccacgguTT
702
36 ± 2
AD-11128
cugacuuguuuacgaaaug
703
cugacuuguuuacgaaaugTT
704
cauuucguaaacaagucagTT
705
33 ± 7
AD-11129
uguuuacgaaauguccaca
706
uguuuacgaaauguccacaTT
707
uguggacauuucguaaacaTT
708
46 ± 8
AD-11130
ccaccgagccagcuugguc
709
ccaccgagccagcuuggucTT
710
gaccaagcuggcucgguggTT
711
51 ± 12
AD-11131
caccgagccagcuuggucc
712
caccgagccagcuugguccTT
713
ggaccaagcuggcucggugTT
714
53 ± 15
AD-11132
caggcaacgugcgugucuc
715
caggcaacgugcgugucucTT
716
gagacacgcacguugccugTT
717
46 ± 6
AD-11133
aacgugcgugucucugcca
718
aacgugcgugucucugccaTT
719
uggcagagacacgcacguuTT
720
59 ± 6
AD-11134
uuaauuuuaacguaacucu
721
uuaauuuuaacguaacucuTT
722
agaguuacguuaaaauuaaTT
723
64 ± 16
AD-11135
uuaacguaacucuuucuau
724
uuaacguaacucuuucuauTT
725
auagaaagaguuacguuaaTT
726
57 ± 6
AD-11136
uaacguaacucuuucuaug
727
uaacguaacucuuucuaugTT
728
cauagaaagaguuacguuaTT
729
72 ± 9
AD-11137
aacguaacucuuucuaugc
730
aacguaacucuuucuaugcTT
731
gcauagaaagaguuacguuTT
732
68 ± 8
AD-11138
guaacucuuucuaugcccg
733
guaacucuuucuaugcccgTT
734
cgggcauagaaagaguuacTT
735
69 ± 10
AD-11139
uaugcccguguaaaguaug
736
uaugcccguguaaaguaugTT
737
cauacuuuacacgggcauaTT
738
102 ± 4
AD-11140
ugcccguguaaaguaugug
739
ugcccguguaaaguaugugTT
740
cacauacuuuacacgggcaTT
741
104 ± 9
AD-11141
ugagcacccgcugacauuu
742
ugagcacccgcugacauuuTT
743
aaaugucagcgggugcucaTT
744
110 ± 25
AD-11142
cacccgcugacauuuccgu
745
cacccgcugacauuuccguTT
746
acggaaaugucagcgggugTT
747
50 ± 4
AD-11143
uuuuagucaggagagugca
748
uuuuagucaggagagugcaTT
749
ugcacucuccugacuaaaaTT
750
93 ± 17
AD-11144
agccaagucauuaaaaugg
751
agccaagucauuaaaauggTT
752
ccauuuuaaugacuuggcuTT
753
62 ± 4
AD-11145
guuggcgacugucaugugg
754
guuggcgacugucauguggTT
755
ccacaugacagucgccaacTT
756
57 ± 4
AD-11146
gcccuuaagggaagcuacu
757
gcccuuaagggaagcuacuTT
758
aguagcuucccuuaagggcTT
759
74 ± 5
AD-11147
gcauaucgcugggcucaac
760
gcauaucgcugggcucaacTT
761
guugagcccagcgauaugcTT
762
61 ± 10
AD-11148
aauaugagcucauuaguaa
763
aaucugagcucauuaguaaTT
764
uuacuaaugagcucauauuTT
765
61 ± 8
AD-11149
gugcccgugucgguucuuc
766
gugcccgugucgguucuucTT
767
gaagaaccgacacgggcacTT
768
66 ± 5
AD-11150
aaugaaaccaggguagaau
769
aaugaaaccaggguagaauTT
770
auucuacccugguuucauuTT
771
101 ± 7
AD-11151
cacccagaauguagcaucu
772
cacccagaauguagcaucuTT
773
agaugcuacauucugggugTT
774
98 ± 8
AD-11152
gagcucgggacggauagua
775
gagcucgggacggauaguaTT
776
uacuauccgucccgagcucTT
777
77 ± 2
AD-11153
ugacaacugaaggcaaccu
778
ugacaacugaaggcaaccuTT
779
agguugccuucaguugucaTT
780
86 ± 3
AD-11154
caacguggaccugccuacg
781
caacguggaccugccuacgTT
782
cguaggcagguccacguugTT
783
86 ± 4
AD-11155
gacugacgagagauguaua
784
gacugacgagagauguauaTT
785
uauacaucucucgucagucTT
786
72 ± 2
AD-11156
acgagagauguauauuuaa
787
acgagagauguauauuuaaTT
788
uuaaauauacaucucucguTT
769
63 ± 3
TABLE 2
Sequences and activities of dsRNAs with stabilizing modifications tested for HD gene
expression inhibiting activity
SEQ
SEQ
Remaining HD
Duplex
Sense strand sequence
ID
Antisense strand
ID
gene mRNA
name
(5′-3′)
NO:
sequence (5′-3′)
NO:
(% of controls)
AL-DP-5996
cmumumumagumcmgagaacmcmaaumgTT
790
cmauugguucucgacumaaagTT
791
24 ± 7
AL-DP-5997
gumcmacmaaagaacmcmgumgcmagTT
792
cugcmacgguucuuugugacTT
793
21 ± 5
AL-DP-5998
umcmggagumumcmaacmcmumaagcmcmTT
794
ggcuumagguugaacuccgaTT
795
36 ± 9
AL-DP-5999
gaaaumcmcmumgcmumumumagumcmgaTT
796
ucgacumaaagcmaggauuucTT
797
20 ± 4
AL-DP-6000
umcmcmumgcmumumumagumcmgagaacmTT
798
guucucgacumaaagcmaggaTT
799
22 ± 3
AL-DP-6001
umumagumcmgagaacmcmaaumgaumTT
800
aucmauugguucucgacumaaTT
801
23 ± 7
AL-DP-6002
umagumcmgagaacmcmaaumgaumgTT
802
cmaucmauugguucucgacumaTT
803
20 ± 7
AL-DP-6003
cmumgcmumumumagumcmgagaacmcmaTT
804
ugguucucgacumaaagcmagTT
805
26 ± 4
AL-DP-6004
cmgcmumgcmacmcmgacmcmaaagaaTT
806
uucuuuggucggugcmagcgTT
807
42 ± 7
AL-DP-6005
umgcmumumumagumcmgagaacmcmaaTT
808
uugguucucgacumaaagcmaTT
809
21 ± 8
AL-DP-6006
gaacmumacmaumcmgaumcmaumggaTT
810
uccmaugaucgaugumaguucTT
811
21 ± 6
AL-DP-6007
umgaacmumacmaumcmgaumcmaumggTT
812
ccmaugaucgaugumaguucmaTT
813
21 ± 3
AL-DP-6008
cmaaagaacmcmgumgcmagaumaaTT
814
uumaucugcmacgguucuuugTT
815
21 ± 8
AL-DP-6009
cmcmcmacmumgcmgumgaacmaumumcmaTT
816
ugaauguucmacgcmagugggTT
817
22 ± 4
AL-DP-6010
umumumagumcmgagaacmcmaaumgaTT
818
ucmauugguucucgacumaaaTT
819
31 ± 5
AL-DP-6011
umggaaumgumumcmcmggagaaumcmTT
820
gauucuccggaacmauuccmaTT
821
26 ± 4
AL-DP-6012
cmggagumumcmaacmcmumaagcmcmumTT
822
aggcuumagguugaacuccgTT
823
28 ± 6
AL-DP-6013
umggcmaumumumgaumcmcmaumgagcmTT
824
gcucmauggaucmaaaugccmaTT
825
34 ± 11
AL-DP-6014
umcmumggaaumgumumcmcmggagaaTT
826
uucuccggaacmauuccmagaTT
827
23 ± 7
AL-DP-6015
ggcmumgcmaaaumumumacmagagcmTT
828
gcucugumaaauuugcmagccTT
829
29 ± 5
AL-DP-6016
gcmgumgaacmaumumcmacmagcmcmaTT
830
uggcugugaauguucmacgcTT
831
17 ± 5
AL-DP-6017
umcmcmaggumumumaumgaacmumgacmTT
832
gucmaguucmaumaaaccuggaTT
833
19 ± 5
AL-DP-6018
aggcmaaagumgcmumcmumumaggaTT
834
uccumaagagcmacuuugccuTT
835
22 ± 6
AL-DP-6019
aacmumacmaumcmgaumcmaumggagTT
836
cuccmaugaucgaugumaguuTT
837
59 ± 10
AL-DP-6020
cmaumumggaaumumcmcmumaaaaumcmTT
838
gauuuumaggaauuccmaaugTT
839
19 ± 11
AL-DP-6021
aumcmcmumgcmumumumagumcmgagaaTT
840
uucucgacumaaagcmaggauTT
841
35 ± 9
AL-DP-6022
acmumacmaumcmgaumcmaumggagaTT
842
ucuccmaugaucgaugumaguTT
843
35 ± 18
AL-DP-6023
aaumcmcmumgcmumumumagumcmgagaTT
844
ucucgacumaaagcmaggauuTT
845
26 ± 16
AL-DP-6024
umgumcmcmaggumumumaumgaacmumgTT
846
cmaguucmaumaaaccuggacmaTT
847
16 ± 5
AL-DP-6025
cmumcmggagumumcmaacmcmumaagcmTT
848
gcuumagguugaacuccgagTT
849
24 ± 6
AL-DP-6026
umgaaaumcmcmumgcmumumumagumcmgTT
850
cgacumaaagcmaggauuucmaTT
851
21 ± 6
AL-DP-6027
cmagcmumumgumcmcmaggumumumaumgTT
852
cmaumaaaccuggacmaagcugTT
853
22 ± 6
AL-DP-6028
cmgumgaacmaumumcmacmagcmcmagTT
854
cuggcugugaauguucmacgTT
855
33 ± 11
AL-DP-6029
cmumggcmumcmgcmaumggumcmgacmaTT
856
ugucgaccmaugcgagccmagTT
857
45 ± 15
AL-DP-6030
agcmumumgumcmcmaggumumumaumgaTT
858
ucmaumaaaccuggacmaagcuTT
859
75 ± 15
AL-DP-6031
ggcmaaagumgcmumcmumumaggagTT
860
cuccumaagagcmacuuugccTT
861
28 ± 10
AL-DP-6032
gaumcmaumumggaaumumcmcmumaaaTT
862
uuumaggaauuccmaaugaucTT
863
25 ± 10
AL-DP-6033
cmacmumgcmgumgaacmaumumcmacmaTT
864
ugugaauguucmacgcmagugTT
865
24 ± 3
AL-DP-6034
gumcmgagaacmcmaaumgaumggcmTT
866
gccmaucmauugguucucgacTT
867
20 ± 1
AL-DP-6035
cmumumgumcmcmaggumumumaumgaacmTT
868
guucmaumaaaccuggacmaagTT
869
28 ± 9
AL-DP-6036
umgumgaumggcmaumcmaumggcmcmaTT
870
uggccmaugaugccmaucmacmaTT
871
50 ± 14
AL-DP-6037
cmacmaaagaacmcmgumgcmagaumTT
872
aucugcmacgguucuuugugTT
873
20 ± 5
dsRNA Synthesis
Source of Reagents
Where the source of a reagent is not specifically given herein, such reagent may be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.
siRNA Synthesis
Single-stranded RNAs were produced by solid phase synthesis on a scale of 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass (CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support. RNA and RNA containing 2′-O-methyl nucleotides were generated by solid phase synthesis employing the corresponding phosphoramidites and 2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg, Germany). These building blocks were incorporated at selected sites within the sequence of the oligoribonucleotide chain using standard nucleoside phosphoramidite chemistry such as described in Current protocols in nucleic acid chemistry, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioate linkages were introduced by replacement of the iodine oxidizer solution with a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further ancillary reagents were obtained from Mallinckrodt Baker (Griesheim, Germany).
Deprotection and purification of the crude oligoribonucleotides by anion exchange HPLC were carried out according to established procedures. Yields and concentrations were determined by UV absorption of a solution of the respective RNA at a wavelength of 260 nm using a spectral photometer (DU 640B, Beckman Coulter GmbH, Unterschleiβheim, Germany). Double stranded RNA was generated by mixing an equimolar solution of complementary strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3 minutes and cooled to room temperature over a period of 3-4 hours. The annealed RNA solution was stored at −20° C. until use.
For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referred to as -Chol or -sChol, depending on whether the link to the cholesteryl group is effected via a phosphodiester or a phosporothioate diester group), an appropriately modified solid support was used for RNA synthesis. The modified solid support was prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
A 4.7 M aqueous solution of sodium hydroxide (50 mL) was added into a stirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred at room temperature until completion of the reaction was ascertained by TLC. After 19 h the solution was partitioned with dichloromethane (3×100 mL). The organic layer was dried with anhydrous sodium sulfate, filtered and evaporated. The residue was distilled to afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionic acid ethyl ester AB
Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was dissolved in dichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to the solution at 0° C. It was then followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was brought to room temperature and stirred further for 6 h. Completion of the reaction was ascertained by TLC. The reaction mixture was concentrated under vacuum and ethyl acetate was added to precipitate diisopropyl urea. The suspension was filtered. The filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. The combined organic layer was dried over sodium sulfate and concentrated to give the crude product which was purified by column chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) was dissolved in 20% piperidine in dimethylformamide at 0° C. The solution was continued stirring for 1 h. The reaction mixture was concentrated under vacuum, water was added to the residue, and the product was extracted with ethyl acetate. The crude product was purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester AD
The hydrochloride salt of 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane. The suspension was cooled to 0° C. on ice. To the suspension diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) was added. The reaction mixture was stirred overnight. The reaction mixture was diluted with dichloromethane and washed with 10% hydrochloric acid. The product was purified by flash chromatography (10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester AE
Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL of dry toluene. The mixture was cooled to 0° C. on ice and 5 g (6.6 mmol) of diester AD was added slowly with stirring within 20 mins. The temperature was kept below 5° C. during the addition. The stirring was continued for 30 mins at 0° C. and 1 mL of glacial acetic acid was added, immediately followed by 4 g of NaH2PO4.H2O in 40 mL of water The resultant mixture was extracted twice with 100 mL of dichloromethane each and the combined organic extracts were washed twice with 10 mL of phosphate buffer each, dried, and evaporated to dryness. The residue was dissolved in 60 mL of toluene, cooled to 0° C. and extracted with three 50 mL portions of cold pH 9.5 carbonate buffer. The aqueous extracts were adjusted to pH 3 with phosphoric acid, and extracted with five 40 mL portions of chloroform which were combined, dried and evaporated to dryness. The residue was purified by column chromatography using 25% ethylacetate/hexane to afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
Methanol (2 mL) was added dropwise over a period of 1 h to a refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring was continued at reflux temperature for 1 h. After cooling to room temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted with ethylacetate (3×40 mL). The combined ethylacetate layer was dried over anhydrous sodium sulfate and concentrated under vacuum to yield the product which was purified by column chromatography (10% MeOH/CHCl3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamic acid 17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with pyridine (2×5 mL) in vacuo. Anhydrous pyridine (10 mL) and 4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with stirring. The reaction was carried out at room temperature overnight. The reaction was quenched by the addition of methanol. The reaction mixture was concentrated under vacuum and to the residue dichloromethane (50 mL) was added. The organic layer was washed with 1M aqueous sodium bicarbonate. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residual pyridine was removed by evaporating with toluene. The crude product was purified by column chromatography (2% MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl3) (1.75 g, 95%).
Succinic acid mono-(4[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl 2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl) ester AH
Compound AG (1.0 g, 1.05 mmol) was mixed with succinic anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40° C. overnight. The mixture was dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at room temperature under argon atmosphere for 16 h. It was then diluted with dichloromethane (40 mL) and washed with ice cold aqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness. The residue was used as such for the next step.
Cholesterol Derivatised CPG AI
Succinate AH (0.254 g, 0.242 mmol) was dissolved in a mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL), 2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively. To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol) in acetonitrile (0.6 ml) was added. The reaction mixture turned bright orange in color. The solution was agitated briefly using a wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The CPG was filtered through a sintered funnel and washed with acetonitrilé, dichloromethane and ether successively. Unreacted amino groups were masked using acetic anhydride/pyridine. The achieved loading of the CPG was measured by taking UV measurement (37 mM/g).
The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamide group (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivative group (herein referred to as “5′-Chol-”) was performed as described in WO 2004/065601, except that, for the cholesteryl derivative, the oxidation step was performed using the Beaucage reagent in order to introduce a phosphorothioate linkage at the 5′-end of the nucleic acid oligomer.
Nucleic acid sequences are represented below using standard nomenclature, and specifically the abbreviations of Table 3.
TABLE 3
Abbreviations of nucleotide monomers used in nucleic
acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are
mutually linked by 5′-3′-phosphodiester bonds.
Abbreviationa
Nucleotide(s)
A, a
2′-deoxy-adenosine-5′-phosphate,
adenosine-5′-phosphate
C, c
2′-deoxy-cytidine-5′-phosphate,
cytidine-5′-phosphate
G, g
2′-deoxy-guanosine-5′-phosphate,
guanosine-5′-phosphate
T, t
2′-deoxy-thymidine-5′-phosphate,
thymidine-5′-phosphate
U, u
2′-deoxy-uridine-5′-phosphate,
uridine-5′-phosphate
N, n
any 2′-deoxy-nucleotide/nucleotide
(G, A, C, or T, g, a, c or u)
am
2′-O-methyladenosine-5′-phosphate
cm
2′-O-methylcytidine-5′-phosphate
gm
2′-O-methylguanosine-5′-phosphate
tm
2′-O-methyl-thymidine-5′-phosphate
um
2′-O-methyluridine-5′-phosphate
Af
2′-fluoro-2′-deoxy-adenosine-5′-phosphate
Cf
2′-fluoro-2′-deoxy-cytidine-5′-phosphate
Gf
2′-fluoro-2′-deoxy-guanosine-5′-phosphate
Tf
2′-fluoro-2′-deoxy-thymidine-5′-phosphate
Uf
2′-fluoro-2′-deoxy-uridine-5′-phosphate
A, C, G, T, U, a,
underlined: nucleoside-5′-phosphorothioate
c, g, t, u
am, cm, gm, tm,
underlined: 2-O-methyl-nucleoside-5′-
um
phosphorothioate
acapital letters represent 2′-deoxyribonucleotides (DNA), lower case letters represent ribonucleotides (RNA)
Screen of HD dsRNAs Against Endogenous Human HD mRNA Expression in HeLa Cells
HeLa cells were obtained from American Type Culture Collection (Rockville, Md.) and cultured in Ham's F12 (Biochrom AG, Berlin, Germany) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
For transfection with siRNA, HeLa cells were seeded at a density of 2.0×104 cells/well in 96-well plates and transfected directly. Transfection of siRNA (30 nM for single dose screen) was carried out with oligofectamine (Invitrogen GmbH, Karlsruhe, Germany) as described by the manufacturer. For dose-response curves, siRNA concentrations ranged from 30 nM to 14 pM in 3-fold dilutions.
24 hours after transfection, HeLa cells were lysed and Huntingtin mRNA levels were quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA) according to the protocol. Huntingtin mRNA levels were normalized to GAPDH mRNA. For each siRNA, four individual datapoints were collected. An siRNA duplex unrelated to the HD gene was used as a control (‘VEGF ctrl’). The activity of a given HD-specific siRNA duplex was expressed as percent HD mRNA concentration in treated cells relative to huntingtin mRNA concentration in cells treated with the control siRNA duplex.
Table 1 provides the results from four independent experiments of the in vitro HeLa screen where the siRNAs, the sequences of which are given in Table 1, were tested at a single dose of 30 nM. The percentage of HD mRNA remaining in treated cells compared to controls, ±standard deviation, is indicated in the rightmost column of Table 1. FIG. 1 provides a graph of the results from two independent experiments of the in vitro HeLa screen where siRNAs, the sequences of which are given in Table 2, were tested at a single dose of 30 nM. In Table 2, duplex names are given as AL-DP-xxxx whereas the same duplex in FIG. 1 is indicated by ‘xxxx’ only. For instance, AL-DP-5997 in Table 2 corresponds to ‘5997’ in FIG. 1. Again, the percentage of HD mRNA remaining in treated cells compared to controls, ±standard deviation, is indicated in the rightmost column of Table 2. A number of siRNAs at 30 nM were effective at reducing HD mRNA levels by more than 70% in HeLa cells.
Table 4 provides the IC50, IC80 and maximum inhibition values from two to five independent experiments for 25 selected siRNAs. Several siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032, indicated by *) were particularly potent in this experimental paradigm, and exhibited IC50 values between 10 and 130 pM.
TABLE 4
IC-50 mean
IC-80 mean
max. inhib.
Duplex name
[nM] ± SD
[nM] ± SD
mean[%] ± SD
AL-DP-5996
1.6 ± 1.2
22 ± 9
79 ± 6
AL-DP-5997*
0.05 ± 0.02
2 ± 1
86 ± 5
AL-DP-5999
0.3 ± 0.3
8 ± 4
82 ± 4
AL-DP-6000*
0.1 ± 0.1
5 ± 3
80 ± 2
AL-DP-6001*
0.1 ± 0.1
3 ± 1
83 ± 1
AL-DP-6002
0.3 ± 0.2
9 ± 4
78 ± 3
AL-DP-6003
0.3 ± 0.2
3 ± 2
83 ± 3
AL-DP-6005
0.3 ± 0.3
9 ± 9
77 ± 7
AL-DP-6006
0.5 ± 0.1
8 ± 5
81 ± 2
AL-DP-6007
0.2 ± 0.1
5 ± 3
77 ± 8
AL-DP-6008
0.16
13.56
75
AL-DP-6014*
0.1 ± 0.1
6 ± 3
81 ± 6
AL-DP-6016
0.2 ± 0.3
8 ± 10
81 ± 8
AL-DP-6017
0.4 ± 0.1
5 ± 4
82 ± 2
AL-DP-6018
0.2 ± 0.04
7 ± 1
81 ± 3
AL-DP-6020*
0.009 ± 0.01
1 ± 1
88 ± 5
AL-DP-6024
0.3 ± 0.1
6 ± 4
88 ± 1
AL-DP-6025
0.3 ± 0.3
11 ± 8
80 ± 1
AL-DP-6026
0.2 ± 0.2
5 ± 4
81 ± 4
AL-DP-6027
0.5 ± 0.1
8 ± 6
81 ± 2
AL-DP-6032*
0.016 ± 0.01
3 ± 5
87 ± 7
AL-DP-6033
0.3 ± 0.2
6 ± 2
78 ± 3
AL-DP-6034
0.7 ± 0.03
10 ± 3
77 ± 4
AL-DP-6035
0.8 ± 0.9
7 ± 5
80 ± 11
AL-DP-6037
0.2 ± 0.1
8 ± 7
79 ± 6
Screen of Selected HD dsRNAs Against Endogenous HD mRNA Expression in Neuroscreen and U87MG Cells
Neuroscreen cells (a PC12 sub-clone) were obtained from Cellomics (Pittsburgh, Pa.) and cultured in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented to contain 5% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany), 10% DHS (Biochrom AG, Berlin, Germany), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany) and 2 mM L-glutamine (Biochrom AG, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
U87MG cells were obtained from American Type Culture Collection (Rockville, Md.) and cultured in Ham's F12 (Biochrom AG, Berlin, Germany) supplemented to contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany), Penicillin 100 U/ml, Streptomycin 100 μg/ml (Biochrom AG, Berlin, Germany) at 37° C. in an atmosphere with 5% CO2 in a humidified incubator (Heraeus HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
Transfection of Neuroscreen and U87MG cells with six selected siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032), and quantitation of Huntingtin and GAPDH mRNA levels with the Quantigene Explore Kit were performed in a similar manner to that described for HeLa cells.
IC50 values are provided in Table 5. In both Neuroscreen (rat) and U87MG (human) cells, IC50s were higher than in HeLa cells, in general. Of the six siRNAs tested, AL-DP-6014 was significantly less potent than the other five siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6020 and AL-DP-6032) against HD mRNA in Neuroscreen cells, whereas AL-DP-6000 was significantly less potent than the other five siRNAs (AL-DP-5997, AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032) against HD mRNA in U87MG cells.
TABLE 5
Neuroscreen IC50
U87MG IC50
Duplex name
mean [nM] +/− SD
mean [nM]
AL-DP-5997
6 ± 2.8
2.7
AL-DP-6000
11.7 ± 10
98
AL-DP-6001
18
0.28
AL-DP-6014
264 ± 180
0.47
AL-DP-6020
1.42 ± 0.2
0.17
AL-DP-6032
4.2 ± 2.2
0.49
dsRNAs Targeting HD Reduce Endogenous HD Protein in HeLa Cells
Hela cells were cultured and transfected as previously described with 100 nM of the indicated siRNAs, including six siRNAs against HD (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032) and one control unrelated siRNA (‘ctrl’). 48 hours post-transfection, the cells were harvested and lysed. Proteins in the lysates were separated on an 8% denaturing PAG. Huntingtin and β-actin were detected by standard western blot protocols using antibodies that bind to the proteins. For Huntingtin detection, the membrane was probed with a mouse anti-huntingtin protein monoclonal antibody (Chemicon, U.K.) followed by a horseradish peroxidase-coupled goat anti-mouse secondary antibody (Santa Cruz Biotechnology, California). β-actin was detected by anti-actin goat polyclonal IgG (Santa Cruz, Calif.) followed by a donkey anti-goat Ig-HRP secondary antibody (Santa Cruz, Calif.).
FIG. 2 provides the results. AL-DP-5997 (‘5997'), AL-DP-6000 (‘6000’), AL-DP-6001 (‘6001’), AL-DP-6014 (‘6014’), AL-DP-6020 (‘6020’) and AL-DP-6032 (‘6032’), a decreased the level of Huntingtin protein relative to the control protein β-actin, whereas the control unrelated siRNA (‘ctrl’) had no effect on the level of either protein. These results demonstrate that dsRNAs targeting HD effectively reduce not only HD mRNA levels, but also HD protein levels.
Stability in Cerebrospinal Fluid (CSF) of Selected dsRNAs Targeting HD
Six selected siRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001, AL-DP-6014, AL-DP-6020 and AL-DP-6032) were tested for stability at 5 uM over 48 h at 37° C. in calf and swine CSF, as well as in PBS for comparison. The incubations in CSF were stopped at 1, 2, 4, 8, 24 and 48 hours by proteinase digestion, whereas the incubation in PBS was stopped at 0 and 48 hours. Filtered samples were injected onto the IEX-HPLC under denaturing conditions, and percent recovery of each single strand was determined by measuring the area under the corresponding peak, and expressing this area relative to that obtained at 0 hours in PBS. FIG. 3 and Table 6 provide the results. At least 90% of both sense and antisense strands of AL-DP-5997, AL-DP-6000 and AL-DP-6014 were recovered in both calf and swine CSF (Table 6). In contrast, although 92% of the antisense strand of AL-DP-6001 was recovered in calf CSF, only 73% of the antisense strand was recovered in swine CSF. For AL-DP-6020 and AL-DP-6032, at least 19% of the antisense strand was not recoverable in both calf and swine CSF.
TABLE 6
% full length material after 48 hours
calf
swine
AL-DP
sense
antisense
sense
antisense
5997
103
99
95
101
6000
114
101
114
97
6001
100
92
100
73
6014
91
90
90
94
6020
113
68
104
32
6032
95
21
103
81
The following cleavage sites for AL-DP-6020 and AL-DP-6032 were mapped by comparing the calculated theoretical masses of all probable fragments of both strands with the experimental masses found by MALDI-TOF. For the antisense strand of AL-DP-6020, the fragment 5′-gauuuumaggaauuccmaau-cyclic-PO4-3′ (SEQ ID NO: 874) corresponds to 3′-(n-3) based on the calculated mass of 5973.5 Da, and experimental mass of 5973.0 Da. For the antisense strand of AL-DP-6032, the fragment 5′-uumaggaauuccmaaugaucTT-3′ (SEQ ID NO: 875) corresponds to 5′-(n-1) based on the calculated mass of 6355.0 Da, and experimental mass of 6355.6 Da. Given these cleavage sites, 2 new duplexes were designed with additional chemical stabilization that comprises one additional 2′-OMe group (Table 7): AL-DP-7100 (parent is AL-DP-6020) and AL-DP-7101 (parent is AL-DP-6032).
TABLE 7
Sequences and Modifications of Further Stabilized
dsRNAs AL-DP-7100 and AL-DP-7101
SEQ
SEQ
Duplex
Sense strand sequence
ID
Antisense strand
ID
name
(5′-3′)
NO:
sequence (5′-3′)
NO:
Al-DP-
cmaumumggaaumumcmcmumaaaaumcmTT
876
gauuuumaggaauuccmaaumgTT
877
7100
Al-DP-
gaumcmaumumggaaumumcmcmumaaaTT
878
umuumaggaauuccmaaugaucTT
879
7101
Four selected dsRNAs (AL-DP-5997, AL-DP-6000, AL-DP-6001 and AL-DP-7100) were tested for long-term stability at 5 uM over 14 days at 37° C. in rat CSF, as well as in PBS for comparison. The incubations in CSF were carried out for 0, 1, 3, 5, 7, 10, or 14 days whereas the incubation in PBS was carried out for 14 days. Samples were processed as described above. FIG. 4 shows the results. For AL-DP-6000, the 14 day CSF stability timepoint is not available, for technical reasons. All four dsRNAs are highly stable for 10 to 14 days at 37° C. in rat CSF, with ≦30% loss of antisense or sense strands.
Potency of Cholesterol-Conjugated dsRNAs Targeting HD Against Endogenous Human HD mRNA Expression in HeLa Cells
Previous studies [Soutschek et al., 2004] had demonstrated a beneficial effect of cholesterol conjugation on cellular uptake and/or efficacy of siRNA in vivo. We synthesized dsRNAs AL-DP-6982, AL-DP-6983 and AL-DP-7130 (Table 8) which are cholesterol-conjugated versions of AL-DP-5997, AL-DP-6000 and AL-DP-7100, respectively, in order to evaluate their biological activities in vitro and in vivo. Hela cells were cultured and transfected as previously described, with dsRNAs AL-DP-6982, AL-DP-6983, AL-DP-7130, AL-DP-5997, AL-DP-6000, and AL-DP-7100 at concentrations ranging from 30 nM to 14 pM.
TABLE 8
Sequences of Cholesterol-Conjugated dsRNAs AL-DP-6982,
AL-DP-6983 and AL-DP-7130
SEQ
SEQ
Duplex
Sense strand sequence
ID
Antisense strand
ID
name
(5′-3′)
NO:
sequence (5′-3′)
NO:
AL-DP-
gumcmacmaaagaacmcmgumgcmagTT-sChol
880
cugcmacgguucuuugugacTT
881
6982
AL-DP-
umcmcmumgcmumumumagumcmgagaacmTT-sChol
882
guucucgacumaaagcmaggaTT
883
6983
AL-DP-
cmaumumggaaumumcmcmumaanaumcmTT-sChol
884
gauuuumaggaauuccmaaumgTT
885
7130
Note:
‘s’ represents a phosphorothioate bound inbetween T and cholesterol, Chol represents cholesterol-conjugate
24 hours after transfection, HeLa cells were lysed and Huntingtin and GAPDH mRNA levels were quantified as described above. For each siRNA, four individual datapoints were collected. An siRNA duplex unrelated to the HD gene was used as a control. The activity of a given siRNA duplex targeting HD was expressed as percent HD mRNA concentration in treated cells relative to the HD mRNA concentration in cells treated with the control siRNA duplex. XL-fit was used to calculate IC50 values; the mean IC50 values were calculated from three independent determinations, and are shown in Table 9.
TABLE 9
Potency of Cholesterol-Conjugated dsRNAs AL-DP-6982,
AL-DP-6983 and AL-DP-7130 Compared with Unconjugated
dsRNAs AL-DP-5997, AL-DP-6000 and AL-DP-7100 against
endogenous human HD mRNA expression in HeLa cells
Duplex name
IC50 (mean, nM)
AL-DP-5997
0.04
AL-DP-6982
0.73
AL-DP-6000
0.24
AL-DP-6983
14.0
AL-DP-7100
0.03
AL-DP-7130
0.38
The unconjugated dsRNAs exhibited expected (Table 4) potencies in vitro against HD mRNA. The cholesterol-conjugated dsRNAs retain biological activity in vitro against HD mRNA, although the potencies are somewhat reduced compared to the unconjugated parent molecules.
In Vivo Down-Modulation of Endogenous HD mRNA Levels by CNS Administration of Unconjugated or Cholesterol-Conjugated dsRNAs Targeting HD in Rats and Mice
To assess both the in vivo biological activity and distribution of unconjugated or cholesterol-conjugated dsRNAs targeting HD, dsRNAs AL-DP-1997 and AL-DP-1998 (Table 10), based on AL-DP-5997, were synthesized in which the two 2′-deoxy-thymidine-5′-phosphate nucleotides at the 3′-end of the antisense strand (outside of the dsRNA's nucleotide region that targets the HD mRNA) were replaced with 5-bromo-2′-deoxyuridine.
TABLE 10
Sequences of dsRNAs AL-DP-1997 and AL-DP-1998
SEQ
SEQ
Duplex
Sense strand sequence
ID
Antisense strand
ID
name
(5’-3’)
NO:
sequence (5’-3’)
NO:
AL-DP-1997
gumcmacmaaagaacmcmgumgcmagTT
886
cugcmacgguucuuugugacBB
887
AL-DP-1998
gumcmacmaaagaacmcmgumgcmagTT-Chol
888
cugcmacgguucuuugugacBB
889
Note:
‘B’ represents 5-bromo-2’-deoxyuridine, underline designates nucleoside-5’-phosphorothioate, Chol represents cholesterol-conjugate
In rats, 1.3 mg AL-DP-1997 or AL-DP-1998, or phosphate-buffered saline (PBS, vehicle control) was administered by continuous intrastriatal infusion over 7 days. Male Sprague-Dawley rats, approximately 250-300 g body weight, received stereotaxic implantation of 30-gauge infusion cannulae (Plastics One, Roanok, Va.) such that unilateral injections were targeted to the center of the striatum (anteroposterior+0.7 mm, mediolateral+3.0 mm, relative to bregma; dorsoventral 5 mm, relative to skull surface). Mini-osmotic pumps (model 1007D) were primed overnight according to the manufacturer's specifications, implanted subcutaneously, and connected via catheters, to deliver (4 rats per treatment group) PBS, 1.1 mM AL-DP-1997 or 1.1 mM AL-DP-1998 at 0.5 uL/hr over 7 days. At the end of the 7 day infusion period, animals were sacrificed, brains were removed, and ipsilateral striata encompassing the infusion site were flash frozen. Tissue samples of about 5-30 mg each were homogenized by sonication (BANDELIN electronic GmbH & Co. KG, Berlin, Germany) in Tissue and Cell Lysis solution (Epicentre, Madison, Wis.) containing 84 μg/ml Proteinase K (Epicentre, Madison, Wis.). Lysates were then stored at −80° C. For carrying out the bDNA assay, frozen lysates were thawed at room temperature, and Huntingtin and GAPDH mRNA were quantified using the Quantigene Explore Kit according to the manufacturer's instructions. For each tissue sample, the ratio of Huntingtin/GAPDH (normalized Huntingtin mRNA level) was calculated as an average of four determinations. These ratios were then averaged to obtain a group (treatment) average. The unconjugated dsRNA, AL-DP-1997, reduced the normalized Huntingtin mRNA level by 33%, relative to the PBS control group, whereas the cholesterol-conjugated dsRNA, AL-DP-1998, reduced the normalized Huntingtin mRNA level by 26%, relative to the PBS control group. Both reductions were statistically significant (p<0.05, ANOVA with Tukey post-hoc analysis). These results demonstrate that intrastriatal AL-DP-1997 and AL-DP-1998 are efficacious in vivo in down-modulating HD mRNA levels.
With an identical experimental paradigm, AL-DP-5997 and AL-DP-6000 were also found to be effective in vivo in down-modulating HD mRNA levels after intrastriatal infusion with 1.3 mg over 7 days (0.5 uL/hr at 1.1 mM) in rats. AL-DP-5997 and AL-DP-6000 reduced the normalized Huntingtin mRNA levels in striatal tissue by 34% and 36%, respectively, relative to the PBS control group. In addition, AL-DP-5997 and AL-DP-6000 reduced the normalized Huntingtin mRNA levels in cortical tissue by 22% and 26% respectively. These results demonstrate that these unconjugated siRNAs, after intrastriatal infusion, not only down-modulate HD mRNA levels within the striatum, but also in the cortex, another major brain region where neuronal loss occurs in Huntington's disease and which is located further from the infusion site.
In mice, 75 ug AL-DP-1998, or phosphate-buffered saline (PBS, vehicle control) was administered by a 20 minute intrastriatal infusion. Male Balb/c mice, approximately 20-25 g body weight, received unilateral injections of test article that were targeted to the striatum (anteroposterior+0.5 mm, mediolateral+2.0 mm, relative to bregma; dorsoventral 3.5 mm, relative to skull surface). Test articles (1.1 mM) were injected (4 animals per test article) at 0.25 uL/min. using pre-filled, pump-regulated Hamilton micro-syringes connected to a 33 gauge needle. Approximately 72 hours following the injection, animals were sacrificed, brains were removed, and ipsilateral striata encompassing the infusion site were dissected and flash frozen. As described above for rat tissue samples, mouse tissue samples were lysed, and Huntingtin and GAPDH mRNA levels quantified. For each tissue sample, the ratio of Huntingtin/GAPDH (normalized Huntingtin mRNA level) was calculated as an average of four determinations. These ratios were then averaged to obtain a group (treatment) average. The cholesterol-conjugated dsRNA, AL-DP-1998, reduced the normalized Huntingtin mRNA level by 33%, relative to the PBS control group, which was statistically significant (p<0.05, ANOVA with Tukey post-hoc analysis). These results further confirm that AL-DP-1998 is efficacious in vivo in down-modulating HD mRNA levels. In addition, these results demonstrate that a total intrastriatal dose of AL-DP-1998 as low as 75 ug resulted in significant down-modulation of HD mRNA levels.
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13252917
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alynylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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514/ 44.A
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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nasdaq:alny
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Alnylam Pharmaceuticals
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Feb 27th, 2018 12:00AM
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Oct 19th, 2012 12:00AM
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https://www.uspto.gov?id=US09902954-20180227
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Method and medicament for inhibiting the expression of a given gene
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The invention relates to an isolated RNA that mediates RNA interference of an mRNA to which it corresponds and a method of mediating RNA interference of mRNA of a gene in a cell or organism using the isolated RNA.
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9902954
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1. A method for inhibiting expression of a target gene, comprising:
a) introducing into a mammalian cell an isolated double stranded RNA (dsRNA) comprising two complementary oligoribonucleotide strands, wherein a first strand of the dsRNA is complementary to an RNA transcript of at least part of the target gene and a second strand of the dsRNA is complementary to the first strand, wherein the dsRNA is 15 to 21 base pairs in length, and wherein dsRNA is introduced into the mammalian cell at a concentration that is lower by at least one order of magnitude than a concentration that would be required for the first strand of the dsRNA alone as single-stranded oligoribonucleotide to inhibit expression of the target gene; and
b) maintaining the cell produced in step a) with the concentration of dsRNA for a time sufficient to obtain degradation of an RNA transcript of the target gene, thereby inhibiting the expression of the target gene at the concentration of the dsRNA.
2. The method of claim 1, wherein at least one of said strands comprises at least one chemically modified nucleotide.
3. The method of claim 2, wherein said chemically modified nucleotide is a 2′-modified nucleotide.
4. The method of claim 2, wherein said 2′-modified nucleotide is a 2′-methyl substituted nucleotide.
5. The method of claim 2, wherein said 2′-modified nucleotide is a 2′-amino substituted nucleotide.
6. The method of claim 2, wherein said chemically modified nucleotide is a locked nucleotide.
7. The method of claim 1, wherein the dsRNA is enclosed by a micellar structure.
8. The method of claim 7, wherein the micellar structure comprises a liposome.
9. The method of claim 1, wherein the two complementary strands are fully complementary to each other.
10. The method of claim 1, wherein said dsRNA specifically inhibits the expression of the target gene using dsRNA-mediated interference.
11. The method of claim 1, wherein one of the two complementary oligoribonucleotide strands is a sense strand, and wherein the sense strand comprises a 2′-methyl substituted nucleotide.
12. The method of claim 1, wherein one of the two complementary oligoribonucleotide strands is a sense strand, and wherein the sense strand comprises a plurality of 2′-methoxynucleotides.
13. The method of claim 1, wherein the RNA transcript comprises a third strand, and wherein the dsRNA, when introduced into the presence of the third strand, reduces an amount of the third strand, the dsRNA thereby specifically inhibiting expression of the target gene.
14. The method of claim 1, wherein introducing the dsRNA into the mammalian cell comprises introducing a pharmaceutical composition comprising the dsRNA into the mammalian cell.
15. A method for inhibiting expression of a target gene, comprising:
a) introducing into a mammalian cell a pharmaceutical composition comprising an isolated double stranded RNA (dsRNA) comprising two complementary oligoribonucleotide strands, wherein a first strand of the dsRNA is complementary to an RNA transcript of at least part of the target gene and a second strand of the dsRNA is complementary to the first strand, wherein the dsRNA is 15 to 21 base pairs in length, and wherein dsRNA is introduced into the mammalian cell at a concentration that is lower by at least one order of magnitude than a concentration that would be required for the first strand of the dsRNA alone as single-stranded oligoribonucleotide to inhibit expression of the target gene; and
b) maintaining the cell produced in step a) with the concentration of dsRNA for a time sufficient to obtain degradation of an RNA transcript of the target gene, thereby inhibiting the expression of the target gene at the concentration of the dsRNA.
16. The method of claim 15, wherein the pharmaceutical composition introduced comprises a liposome enclosing the dsRNA.
17. The method of claim 2, wherein the at least one chemically modified nucleotide is at the 3′ terminus, the 5′ terminus or combinations thereof, of at least one of the oligoribonucleotide strands.
18. The method of claim 17, wherein the at least one chemically modified nucleotide is at the 3′ terminus.
19. The method of claim 17 or 18, wherein the at least one chemically modified nucleotide is a 2′-methyl substituted nucleotide.
20. The method of claim 1, wherein the mammalian cell is a primate cell.
21. The method of claim 15, wherein the dsRNA comprises at least one chemically modified nucleotide at the 3′ terminus, the 5′ terminus or combinations thereof, of at least one of the oligoribonucleotide strands.
22. The method of claim 21, wherein the at least one chemically modified nucleotide is at the 3′ terminus.
23. The method of claim 21 or 22, wherein the at least one chemically modified nucleotide is a 2′-methyl substituted nucleotide.
24. The method of claim 15, wherein the mammalian cell is a primate cell.
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24
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CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 11/982,325, filed Oct. 31, 2007, which is a continuation of U.S. patent application Ser. No. 10/612,179, filed Jul. 2, 2003, now issued, which is a divisional of U.S. patent application Ser. No. 09/889,802, filed Sep. 17, 2001, now abandoned, which is the National Stage of International Patent Application No. PCT/DE00/00244, filed Jan. 29, 2000, which claims priority to German Patent Application No. DE19903713.2, filed Jan. 30, 1999, and German Patent Application No. DE19956568.6, filed Nov. 24, 1999. The contents of these prior applications are hereby incorporated by reference in their entirety for all purposes.
The invention relates to a medicament and to a use of double-stranded oligoribonucleotides and to a vector encoding them.
Such a method is known from WO 99/32619, which was unpublished at the priority date of the present invention. The known process aims at inhibiting the expression of genes in cells of invertebrates. To this end, the double-stranded oligoribonucleotide must exhibit a sequence which is identical with the target gene and which has a length of at least 50 bases. To achieve efficient inhibition, the identical sequence must be 300 to 1000 base pairs in length. Such an oligoribonucleotide is complicated to prepare.
DE 196 31 919 C2 describes an antisense RNA with specific secondary structures, the antisense RNA being present in the form of a vector encoding it. The antisense RNA takes the form of an RNA molecule which is complementary to regions of the mRNA. Inhibition of the gene expression is caused by binding to these regions. This inhibition can be employed in particular for the diagnosis and/or therapy of diseases, for example tumor diseases or viral infections. —The disadvantage is that the antisense RNA must be introduced into the cell in an amount which is at least as high as the amount of the mRNA. The known antisense methods are not particularly effective.
U.S. Pat. No. 5,712,257 discloses a medicament comprising mismatched double-stranded RNA (dsRNA) and bioactive mismatched fragments of dsRNA in the form of a ternary complex together with a surfactant. The dsRNA used for this purpose consists of synthetic nucleic acid single strands without defined base sequence. The single strands undergo irregular base pairing, also known as “non-Watson-Crick” base pairing, giving rise to mismatched double strands. The known dsRNA is used to inhibit the amplification of retroviruses such as HIV. Amplification of the virus can be inhibited when non-sequence-specific dsRNA is introduced into the cells. This leads to the induction of interferon, which is intended to inhibit viral amplification. The inhibitory effect, or the activity, of this method is poor.
It is known from Fire, A. et al., NATURE, Vol. 391, pp. 806 that dsRNA whose one strand is complementary in segments to a nematode gene to be inhibited inhibits the expression of this gene highly efficiently. It is believed that the particular activity of the dsRNA used in nematode cells is not due to the antisense principle but possibly on catalytic properties of the dsRNA, or enzymes induced by it. —Nothing is mentioned in this paper on the activity of specific dsRNA with regard to inhibiting the gene expression, in particular in mammalian and human cells.
The object of the present invention is to do away with the disadvantages of the prior art. In particular, it is intended to provide as effective as possible a method, medicament or use for the preparation of a medicament, which method, medicament or use is capable of causing particularly effective inhibition of the expression of a given target gene.
This object is achieved by the features of the claims presented here. Advantageous embodiments can be seen from the claims presented here.
In accordance with the method-oriented inventions, it is provided in each case that the region I which is complementary to the target gene exhibits not more than 49 successive nucleotide pairs.
Provided in accordance with the invention are an oligoribonucleotide or a vector encoding therefor. At least segments of the oligoribonucleotide exhibit a defined nucleotide sequence. The defined segment may be limited to the complementary region I. However, it is also possible that all of the double-stranded oligoribonucleotide exhibits a defined nucleotide sequence.
Surprisingly, it has emerged that an effective inhibition of the expression of the target gene can be achieved even when the complementary region I is not more than 49 base pairs in length. The procedure of providing such oligoribonucleotides is less complicated.
In particular, dsRNA with a length of over 50 nucleotide pairs induces certain cellular mechanisms, for example the dsRNA-dependent protein kinase or the 2-5A system, in mammalian and human cells. This leads to the disappearance of the interference effect mediated by the dsRNA which exhibits a defined sequence. As a consequence, protein biosynthesis in the cell is blocked. The present invention overcomes this disadvantage in particular.
Furthermore, the uptake of dsRNA with short chain lengths into the cell or into the nucleus is facilitated markedly over longer-chain dsRNAs.
It has proved advantageous for the dsRNA or the vector to be present packaged into micellar structures, preferably in liposomes. The dsRNA or the vector can likewise be enclosed in viral natural capsids or in chemically or enzymatically produced artificial capsids or structures derived therefrom. The abovementioned features make it possible to introduce the dsRNA or the vector into given target cells.
In a further aspect, the dsRNA has 10 to 1000, preferably 15 to 49, base pairs. Thus, the dsRNA can be longer than the region I, which is complementary to the target gene. The complementary region I can be located at the terminus or inserted into the dsRNA. Such dsRNA or a vector provided for coding the same can be produced synthetically or enzymatically by customary methods.
The gene to be inhibited is expediently expressed in eukaryotic cells. The target gene can be selected from the following group: oncogene, cytokin gene, Id protein gene, developmental gene, prion gene. It can also be expressed in pathogenic organisms, preferably in plasmodia. It can be part of a virus or viroid which is preferably pathogenic to humans. —The method proposed makes it possible to produce compositions for the therapy of genetically determined diseases, for example cancer, viral diseases or Alzheimer's disease.
The virus or viroid can also be a virus or viroid which is pathogenic to animals or plant-pathogenic. In this case, the method according to the invention also permits the provision of compositions for treating animal or plant diseases.
In a further aspect, segments of the dsRNA are designed as double-stranded. A region II which is complementary within the double-stranded structure is formed by two separate RNA single strands or by autocomplementary regions of a topologically closed RNA single strand which is preferably in circular form.
The ends of the dsRNA can be modified to counteract degradation in the cell or dissociation into the single strands. Dissociation takes place in particular when low concentrations or short chain lengths are used. To inhibit dissociation in a particularly effective fashion, the cohesion of the complementary region II, which is caused by the nucleotide pairs, can be increased by at least one, preferably two, further chemical linkage(s). —A dsRNA according to the invention whose dissociation is reduced exhibits greater stability to enzymatic and chemical degradation in the cell or in the organism.
The complementary region II can be formed by autocomplementary regions of an RNA hairpin loop, in particular when using a vector according to the invention. To afford protection from degradation, it is expedient for the nucleotides to be chemically modified in the loop region between the double-stranded structure.
The chemical linkage is expediently formed by a covalent or ionic bond, a hydrogen bond, hydrophobic interactions, preferably van-der-Waals or stacking interactions, or by metal-ion coordination. In an especially advantageous aspect, it can be formed at at least one, preferably both, end(s) of the complementary region II.
It has furthermore proved to be advantageous for the chemical linkage to be formed by one or more linkage groups, the linkage groups preferably being poly (oxyphosphinicooxy-1,3-propanediol) and/or poly-ethylene glycol chains. The chemical linkage can also be formed by purine analogs used in place of purines in the complementary regions II. It is also advantageous for the chemical linkage to be formed by azabenzene units introduced into the complementary regions II. Moreover, it can be formed by branched nucleotide analogs used in place of nucleotides in the complementary regions II.
It has proved expedient to use at least one of the following groups for generating the chemical linkage: methylene blue; bifunctional groups, preferably bis(2-chloroethyl)amine; N-acetyl-N′-(p-glyoxyl-benzoyl)cystamine; 4-thiouracil; psoralene. The chemical linkage, can furthermore be formed by thiophosphoryl groups provided at the ends of the double-stranded region. The chemical linkage at the ends of the double-stranded region is preferably formed by triple-helix bonds.
The chemical linkage can expediently be induced by ultraviolet light.
The nucleotides of the dsRNA can be modified. This counteracts the activation, in the cell, of a double-stranded-RNA-dependent protein kinase, PKR. Advantageously, at least one 2′-hydroxyl group of the nucleotides of the dsRNA in the complementary region II is replaced by a chemical group, preferably a 2′-amino or a 2′-methyl group. At least one nucleotide in at least one strand of the complementary region II can also be a locked nucleotide with a sugar ring which is chemically modified, preferably by a 2′-O,4′-C methylene bridge. Advantageously, several nucleotides are locked nucleotides.
A further especially advantageous embodiment provides that the dsRNA or the vector is bound to, associated with or surrounded by, at least one viral coat protein which originates from a virus, is derived therefrom or has been prepared synthetically. The coat protein can be derived from polyomavirus. The coat protein can contain the polyomavirus virus protein 1 (VP1) and/or virus protein 2 (VP2). The use of such coat proteins is known from, for example, DE 196 18 797 A1, whose disclosure is herewith incorporated. —The abovementioned features considerably facilitate the introduction of the dsRNA or of the vector into the cell.
When a capsid or capsid-type structure is formed from the coat protein, one side preferably faces the interior of the capsid or capsid-type structure. The construct formed is particularly stable.
The dsRNA can be complementary to the primary or processed RNA transcript of the target gene. —The cell can be a vertebrate cell or a human cell.
At least two dsRNAs which differ from each other or at least one vector encoding them can be introduced into the cell, where at least segments of one strand of each dsRNA are complementary to in each case one of at least two different target genes. This makes it possible simultaneously to inhibit the expression of at least two different target genes. In order to suppress, in the cell, the expression of a double-stranded-RNA-dependent protein kinase, PKR, one of the target genes is advantageously the PKR gene. This allows effective suppression of the PKR activity in the cell.
The invention furthermore provides a medicament with at least one oligoribonucleotide with double-stranded structure (dsRNA) for inhibiting the expression of a given target gene, where one strand of the dsRNA has a region I where at least segments are complementary to the target gene. —Surprisingly, it has emerged that such a dsRNA is suitable as medicament for inhibiting the expression of a given gene in mammalian cells. In comparison with the use of single-stranded oligoribonucleotides, the inhibition is already caused at concentrations which are lower by at least one order of magnitude. The medicament according to the invention is highly effective. Lesser side effects can be expected.
The invention furthermore provides a medicament with at least one vector for coding at least one oligoribonucleotide with double-stranded structure (dsRNA) for inhibiting the expression of a given target gene, where one strand of the dsRNA has a region I where at least segments are complementary to the target gene. —The medicament proposed exhibits the abovementioned advantages. By, using a vector, in particular production costs can be reduced.
In a particularly advantageous embodiment, the complementary region I has not more than 49 successive nucleotide pairs. —Surprisingly, it has emerged that an effective inhibition of the expression of the target gene can be achieved even when the complementary region I is not more than 49 base pairs in length. The procedure of providing such oligoribonucleotides is less complicated.
The invention furthermore provides a use of an oligoribonucleotide with double-stranded structure (dsRNA) for preparing a medicament for inhibiting the expression of a given target gene, where one strand of the dsRNA has a region I where at least segments are complementary to the target gene. —Surprisingly, such a dsRNA is suitable for preparing a medicament for inhibiting the expression of a given gene. Compared with the use of single-stranded oligoribonucleotides, the inhibition is already caused at concentrations which are lower by one order of magnitude when using dsRNA. The use according to the invention thus makes possible the preparation of particularly effective medicaments.
The invention furthermore provides the use of a vector for coding at least one oligoribonucleotide with double-stranded structure (dsRNA) for preparing a medicament for inhibiting the expression of a given target gene, where one strand of the dsRNA has a region I where at least segments are complementary to this target gene. —The use of a vector makes possible a particularly effective gene therapy.
With regard to advantageous embodiments of the medicament and of the use, reference is made to the description of the above features.
Use examples of the invention are illustrated in greater detail hereinbelow with reference to the figures, in which:
FIG. 1 shows the schematic representation of a plasmid for the in vitro, transcription with T7- and SP6-polymerase,
FIG. 2 shows RNA following electrophoresis on an 8% polyacrylamide gel and staining with ethidium bromide,
FIG. 3 shows a representation of radioactive RNA transcripts following electrophoresis on an 8% polyacrylamide gel with 7 M urea by means of an instant imager, and
FIGS. 4a-e show Texas Red and YFP fluorescence in murine fibroblasts.
USE EXAMPLE 1
The inhibition of transcription was detected by means of sequence homologous dsRNA in an in vitro transcription system with a nuclear extract from human HeLa cells. The DNA template for this experiment was plasmid pCMV 1200 which had been linearized by means of BamHI.
Generation of the Template Plasmids:
The plasmid shown in FIG. 1 was constructed for use in the enzymatic synthesis of the dsRNA. To this end, a polymerase chain reaction (PCR) with the “positive control DNA” of the HelaScribe® Nuclear Extract in vitro transcription kit by Promega, Madison, USA, as DNA template was first carried out. One of the primers used contained the sequence of an EcoRI cleavage site and of the T7 RNA polymerase promoter as shown in sequence listing No. 1. The other primer contained the sequence of a BamHI cleavage site and of the SP6 RNA polymerase promoter as shown in sequence listing No. 2. In addition, the two primers had, at the 3′ ends, regions which were identical with or complementary to the DNA template. The PCR was carried out by means of the “Taq PCR Core Kits” by Qiagen, Hilden, Germany, following the manufacturer's instructions. 1.5 mM MgCl2, in each case 200 μM dNTP, in each case 0.5 μM primer, 2.5 U Taq DNA polymerase and approximately 100 ng of “positive control DNA” were employed as template in PCR buffer in a volume of 100 μl. After initial denaturation of the template DNA by heating for 5 minutes at 94° C., amplification was carried out in 30 cycles of denaturation for in each case 60 seconds at 94° C., annealing for 60 seconds at 5° C. below the calculated melting point of the primers and polymerization for 1.5-2 minutes at 72° C. After a final polymerization of 5 minutes at 72° C., 5 μl of the reaction were analyzed by agarose-gel electrophoresis. The length of the DNA fragment amplified thus was 400 base pairs, 340 base pairs corresponding to the “positive control DNA”. The PCR product was purified, hydrolyzed with EcoRI and BamHI and, after repurification, employed in the ligation together with a pUC 18 vector which had also been hydrolyzed by EcoRI and BamHI. E. coli XL1-blue was then transformed. The plasmid obtained (pCMV5) carries a DNA fragment whose 5′ end is flanked by the T7 promoter and whose 3′ end is flanked by the SP6 promoter. By linearizing the plasmid with BamHI, it can be employed in vitro with the T7-RNA polymerase for the run-off transcription of a single-stranded RNA which is 340 nucleotides in length and shown in sequence listing No. 3. If the plasmid is linearized with EcoRI, it can be employed for the run-off transcription with SP6 RNA polymerase, giving rise to the complementary strand. In accordance with the method outlined hereinabove, an RNA 23 nucleotides in length was also synthesized. To this end, a DNA shown in sequence listing No. 4 was ligated with the pUC18 vector via the EcoRI and BamHI cleavage sites.
Plasmid pCMV 1200 was constructed as DNA template for the in-vitro transcription with HeLa nuclear extract. To this end, a 1 191 by EcoRI/BamHI fragment of the positive control DNA contained in the HeLaScribe® Nuclear Extract in vitro transcription kit was amplified by means of PCR. The amplified fragment encompasses the 828 by “immediate early” CMV promoter and a 363 by transcribable DNA fragment. The PCR product was ligated to the vector pGEM-T via “T-overhang” ligation. A BamHI cleavage site is located at the 5′ end of the fragment. The plasmid was linearized by hydrolysis with BamHI and used as template in the run-off transcription.
In-Vitro Transcription of the Complementary Single Strands:
pCMV5 plasmid DNA was linearized with EcoRI or BamHI. It was used as DNA template for an in-vitro transcription of the complementary RNA single strands with SP6 and T7 RNA polymerase, respectively. The “Riboprobe in vitro Transcription” system by Promega, Madison, USA, was employed for this purpose. Following the manufacturer's instructions, 2 μg of linearized plasmid DNA were incubated in 100 μl of transcription buffer and 40 U T7 or SP6 RNA polymerase for 5-6 hours at 37° C. The DNA template was subsequently degraded by addition of 2.5 μl of RNase-free DNase RQ1 and incubation for 30 minutes at 37° C. The transcription reaction was made up to 300 μl with H2O and purified by phenol extraction. The RNA was precipitated by addition of 150 ∞l of 7 M ammonium acatate [sic] and 1 125 μl of ethanol and stored at −65° C. until used for the hybridization.
Generation of the RNA Double Strands:
For the hybridization, 500 μl of the single-stranded RNA which had been stored in ethanol and precipitated were spun down. The resulting pellet was dried and taken up in 30 μl of PIPES buffer, pH 6.4 in the presence of 80% formamide, 400 mM NaCl and 1 mM EDTA. In each case 15 μl of the complementary single strands were combined and heated for 10 minutes at 85° C. The reactions were subsequently incubated overnight at 50° C. and cooled to room temperature.
Only approximately equimolar amounts of the two single strands were employed in the hybridization. This is why the dsRNA preparations contained single-stranded RNA (ssRNA) as contaminant. In order to remove these ssRNA contaminants, the reactions were treated, after hybridization, with the single-strand-specific ribonucleases bovine pancreatic RNase A and Aspergillus oryzae RNase T1. RNase A is an endoribonuclease which is specific for pyrimidines. RNase T1 is an endoribonuclease which preferentially cleaves at the 3′ side of guanosines. dsRNA is no substrate for these ribonucleases. For the RNase treatment, the reactions in 300 μl of Tris, pH 7.4, 300 mM NaCl and 5 mM EDTA were treated with 1.2 μl of RNaseA at a concentration of 10 mg/ml and 2 μl of RNaSeT1 at a concentration of 290 μg/ml. The reactions were incubated for 1.5 hours at 30° C. Thereupon, the RNases were denatured by addition of 5 μl of proteinase K at a concentration of 20 mg/ml and 10 μl of 20% SDS and incubation for 30 minutes at 37° C. The dSRNA was purified by phenol extraction and precipitated with ethanol. To verify the completeness of the RNase digestion, two control reactions were treated with ssRNA analogously to the hybridization reactions.
The dried pellet was taken up in 15 μl of TE buffer, pH 6.5, and subjected to native polyacrylamide gel electrophoresis on an 8% gel. The acrylamide gel was subsequently stained in an ethidium bromide solution and washed in a water bath. FIG. 2 shows the RNA which had been visualized in a UV transilluminator. The sense RNA which had been applied to lane 1 and the antisense RNA which had been applied to lane 2 showed a different migration behavior under the chosen conditions than the dsRNA of the hybridization reaction which had been applied to lane 3. The RNase-treated sense RNA and antisense RNA which had been applied to lanes 4 and 5, respectively, produced no visible band. This shows that the single-stranded RNAs had been degraded completely. The RNase-treated dsRNA of the hybridization reaction which had been applied to lane 6 is resistant to RNase treatment. The band which migrates faster in the native gel in comparison with the dsRNA applied to lane 3 results from dsRNA which is free from ssRNA. In addition to the dominant main band, weaker bands which migrate faster are observed after the RNase treatment.
In-Vitro Transcription Test with Human Nuclear Extract:
Using the HeLaScribe® Nuclear Extract in vitro transcription kit by Promega, Madison, USA, the transcription efficiency of the abovementioned DNA fragment which is present in plasmid pCMV 1200 and homologous to the “positive control DNA” was determined in the presence of the dsRNA (dsRNA-CMV5) with sequence homology. Also, the effect of the dsRNA without sequence homology, which corresponds to the yellow fluorescent protein (YFP) gene (dsRNA-YRP), was studied. This dsRNA had been generated analogously to the dsRNA with sequence homology. The sequence of a strand of this dsRNA can be found in sequence listing No. 5. Plasmid pCMV 1200 was used as template for the run-off transcription. It carries the “immediate early” cytomegalovirus promoter which is recognized by the eukaryotic RNA polymerase II, and a transcribable DNA fragment. Transcription was carried out by means of the HeLa nuclear extract, which contains all the proteins which are necessary for transcription. By addition of [●-32P] rGTP to the transcription reaction, radiolabeled transcript was obtained. The [●-32P] rGTP used had a specific activity of 400 Ci/mmol, 10 mCi/ml. 3 mM MgCl2, in each case 400 μM rATP, rCTP, rUTP, 16 μM rGTP, 0.4 μM [●-32P] rGTP and depending on the experiment 1 fmol of linearized plasmid DNA and various amounts of dsRNA in transcription buffer were employed per reaction. Each batch was made up to a volume of 8.5 μl with H2O. The reactions were mixed carefully. To start the transcription, 4 U HeLa nuclear extract in a volume of 4 μl were added and incubated for 60 minutes at 30° C. The reaction was stopped by addition of 87.5 μl of quench mix which had been warmed to 30° C. To remove the proteins, the reactions were treated with 100 μl of phenol/chloroform/isoamyl alcohol (25:24:1 v/v/v) saturated with TE buffer, pH 5.0, and the reactions were mixed vigorously for 1 minute. For phase separation, the reactions were spun for approximately 1 minute at 12 000 rpm and the top phase was transferred into a fresh reaction vessel. Each reaction was treated with 250 μl of ethanol. The reactions were mixed thoroughly and incubated for at least 15 minutes on dry ice/methanol. To precipitate the RNA, the reactions were spun for 20 minutes at 12 000 rpm and 40° C. The supernatant was discarded. The pellet was dried in vacuo for 15 minutes and resuspended in 10 μl of H2O. Each reaction was treated with 10 μl of denaturing loading buffer. The free GTP was separated from the transcript formed by means of denaturing polyacrylamide gel electrophoresis on an 8% gel with 7 M urea. The RNA transcripts formed upon transcription with HeLa nuclear extract, in denaturing loading buffer, were heated for 10 minutes at 90° C. and 10 μl aliquots were applied immediately to the freshly washed pockets. The electrophoresis was run at 40 mA. The amount of the radioactive ssRNA formed upon transcription was analyzed after electrophoresis with the aid of an Instant Imager.
FIG. 3 shows the radioactive RNA from a representative test, shown by means of the Instant Imager. Samples obtained from the following transcription reactions were applied:
Lane 1: without template DNA, without dsRNA;
Lane 1: 50 ng of template DNA, without dsRNA;
Lane 3: 50 ng of template DNA, 0.5 μg of dsRNA YFP;
Lane 4: 50 ng of template DNA, 1.5 μg of dsRNA YFP;
Lane 5: 50 ng of template DNA, 3 μg of dsRNA: YFP;
Land 6: 50 ng of template DNA, 5 μg of dsRNA YFP;
Lane 7: without template DNA, 1.5 dsRNA YFP;
Lane 8: 50 ng of template DNA, without dsRNA;
Lane 9: 50 ng of template DNA, 0.5 μg of dsRNA CMV5;
Lane 10: 50 ng of template DNA, 1.5 μg of dsRNA CMV5;
Lane 11: 50 ng of template DNA, 3 μg of dsRNA CMV5;
Lane 12: 50 ng of template DNA, 5 μg of dsRNA CMV5;
It emerged that the amount of transcript was reduced markedly in the presence of dsRNA with sequence homology in comparison with the control reaction without dsRNA and with the reactions with dsRNA YFP without sequence homology. The positive control in lane 2 shows that radioactive transcript was formed upon the in-vitro transcription with HeLa nuclear extract. The reaction is used for comparison with the transcription reactions which had been incubated in the presence of dsRNA. Lanes 3 to 6 show that the addition of non-sequentially-specific dsRNA YFP had no effect on the amount of transcript formed. Lanes 9 to 12 show that the addition of an amount of between 1.5 and 3 μg of sequentially-specific dsRNA CMV5 leads to a reduction in the amount of transcript formed. In order to exclude that the effects observed are based not on the dsRNA but on any contamination which might have been carried along accidentally during the preparation of the dsRNA, a further control was carried out. Single-stranded RNA was transcribed as described above and subsequently subjected to the RNase treatment. It was demonstrated by means of native polyacrylamide gel electrophoresis that the ssRNA had been degraded completely. This reaction was subjected to phenol extraction and ethanol precipitation and subsequently taken up in PE buffer, as were the hybridization reactions. This gave a sample which contained no RNA but had been treated with the same enzymes and buffers as the dsRNA. Lane 8 shows that the addition of this sample had no effect on transcription. The reduction of the transcript upon addition of sequence-specific dsRNA can therefore be ascribed unequivocally to the dsRNA itself. The reduction of the amount of transcript of a gene in the presence of dsRNA in a human transcription system indicates an inhibition of the expression of the gene in question. This effect can be attributed to a novel mechanism caused by the dsRNA.
USE EXAMPLE 2
The test system used for these in-vivo experiments was the murine fibroblast cell line NIH3T3, ATCC CRL-1658. The YFP gene was introduced into the nuclei with the aid of microinjection. Expression of YFP was studied under the effect of simultaneously cotransfected dsRNA with sequence homology. This dsRNA YFP shows homology with the 5′-region of the YFP gene over a length of 315 bp. The nucleotide sequence of a strand of the dsRNA YRP is shown in sequence listing No. 5. Evaluation under the fluorescence microscope was carried out 3 hours after injection with reference to the greenish-yellow fluorescence of the YFP formed.
Construction of the Template Plasmid, and Preparation of the dsRNA:
A plasmid was constructed following the same principle as described in use example 1 to act as template for the production of the YFP dsRNA by means of T7 and SP6 in-vitro transcription. Using the primer Eco_T7_YFP as shown in sequence listing No. 6 and Bam_SP6_YFP as shown in sequence listing No. 7, the desired gene fragment was amplified by PCR and used analogously to the above description for preparing the dsRNA. The dsRNA YFP obtained is identical to the dsRNA used in use example 1 as non-sequence-specific control.
A dsRNA linked chemically at the 3′ end of the RNA as shown in sequence listing No. 8 to the 5′ end of the complementary RNA via a C18 linker group was prepared (L-dsRNA). To this end, synthons modified by disulfide bridges were used. The 3′-terminal synthon is bound to the solid support via the 3′ carbon with an aliphatic linker group via a disulfide bridge. In the 5′-terminal synthon of the complementary oligoribonucleotide which is complementary to the 3′-terminal synthon of the one oligoribonucleotide, the 5′-trityl protecting group is bound via a further aliphatic linker and a disulfide bridge. Following synthesis of the two single strands, removal of the protecting groups and hybridization of the complementary oligoribonucleotides, the thiol groups which form are brought into spatial vicinity. The single strands are linked to each other by oxidation via their aliphatic linkers and a disulfide bridge. This is followed by purification with the aid of HPLC.
Preparation of the Cell Cultures:
The cells were incubated in DMEM supplemented with 4.5 g/l glucose, 10% fetal bovine serum in culture dishes at 37° C. under a 7.5% CO2 atmosphere and passaged before reaching confluence. The cells were detached with trypsin/EDTA. To prepare for microinjection, the cells were transferred into Petri dishes and incubated further until microcolonies formed.
Microinjection:
For the microinjection, the culture dishes were removed from the incubator for approximately 10 minutes. Approximately 50 nuclei were injected singly per reaction within a marked area using the AIS microinjection system from Carl Zeiss, Göttingen, Germany. The cells were subsequently incubated for three more hours. For the microinjection, borosilicate glass capillaries from Hilgenberg GmbH, Malsfeld, Germany, with a diameter of less than 0.5 μm at the tip were prepared. The microinjection was carried out using a micromanipulator from Narishige Scientific Instrument Lab., Tokyo, Japan. The injection time was 0.8 seconds and the pressure was approximately 100 hPa. The transfection was carried out using the plasmid pCDNA YFP, which contains an approximately 800 bP BamHI/EcoRI fragment with the YFP gene in vector pcDNA3. The samples injected into the nuclei contained 0.01 μg/μl of pCDNA-YFP and Texas Red coupled to dextran-70000 in 14 mM NaCl, 3 mM KCl, 10 mM KPO4 [sic], ph 7.5. Approximately 100 pl of RNA with a concentration of 1 μM or, in the case of the L-dsRNA, 375 μM were additionally added.
The cells were studied under a fluorescence microscope with excitation with the light of the excitation wavelength of Texas Red, 568 nm, or of YFP, 488 nm. Individual cells were documented by means of a digital carvers. FIGS. 4a-e show the result for NIH3T3 cells. In the cells shown in FIG. 4a, sense-YFP-ssRNA has been injected, in FIG. 4b antisense-YFP-ssRNA, in FIG. 4c dsRNA-YFP, in FIG. 4d no RNA and in FIG. 4e L-dsRNA.
The field on the left shows in each case the fluorescence of cells with excitation at 568 nm. The fluorescence of the same cells at an excitation of 488 nm is seen on the right. The Texas Red fluorescence of all the cells shown demonstrates that the injection solution had been applied successfully into the nuclei and that cells with successful hits were still alive after three hours. Dead cells no longer showed Texas Red fluorescence.
The right fields of each of FIGS. 4a and 4b show that YFP expression was not visibly inhibited when the single-stranded RNA was injected into the nuclei. The right field of FIG. 4c shows cells whose YFP fluorescence was no longer detectable after the injection of dsRNA-YFP. FIG. 4d shows cells into which no RNA had been injected, as control. The cell shown in FIG. 4e shows YFP fluorescence which can no longer be detected owing to the injection of the L-dsRNA which shows regions with sequence homology to the YFP gene. This result demonstrates that even shorter dsRNAs can be used for specifically inhibiting gene expression in mammals when the double strands are stabilized by chemically linking the single strands.
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13656548
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alynylam pharmaceuticals, inc.
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USA
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B2
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Utility Patent Grant (with pre-grant publication) issued on or after January 2, 2001.
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Open
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Apr 1st, 2022 06:06PM
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Apr 1st, 2022 06:06PM
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Alnylam Pharmaceuticals
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Health Care
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Pharmaceuticals & Biotechnology
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